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Structure and genomic organization of the ipiB and ipiO gene clusters of Phytophthora infestans
Abstract
Two in planta-induced (ipi) genes, designated ipiB and ipiO, of the potato late blight fungus, Phytophthora infestans (Mont.) de Bary, were isolated from a genomic library by a differential hybridization procedure [Pieterse et al., Physiol. Mol. Plant Pathol. (1993a) in press]. Both genes are expressed at high levels in the early phases of the pathogenic interaction of P. infestans with its host plant potato, suggesting that their gene products have a function in the early stages of the infection process. Here, we describe the nucleotide (nt) sequence and genomic organization of ipiB and ipiO. The ipiB gene belongs to a small gene family consisting of at least three genes, designated ipiB1, ipiB2 and ipiB3, which are clustered in a head-to-tail arrangement. The three ipiB genes are highly homologous throughout the coding regions and 5' and 3' flanking regions. The P. infestans genome contains two very similar ipiO genes, ipiO1 and ipiO2, which are closely linked and arranged in an inverted orientation. The ipiB genes encode three novel, highly similar Gly-rich proteins of 301, 343 and 347 amino acids (aa), respectively. The Gly-rich domains of the IPI-B proteins are predominantly composed of two repeats with the core sequences, A/V-G-A-G-L-Y-G-R and G-A-G-Y/V-G-G. The ipiO genes code for two almost identical 152-aa proteins which do not have any homology with sequences present in data libraries. IPI-B, as well as IPI-O, contains putative signal peptides of 20 and 21 aa, respectively, suggesting that they are transported out of the cytoplasm. In the promoter regions of ipiB and ipiO, a 16-nt sequence motif, matching the core sequence, GCTCATTYYNCAWTTT (where N = A or C or G or T; W = A or T; Y = C or T), was found. This sequence motif appears to be present around the transcription start point (tsp) of seven out of eight oomycetous genes for which the tsp have been determined, suggesting that oomycetes have a sequence preference for transcription initiation.
Supplementary resources (5)
Nucleotide Sequence
September 1993
... A number of methods exist for isolating such differentially expressed genes. [63] Judelson et al. [55] isolated a number of in planta induced (ipi ) genes by differential screening of a P. infestans genomic DNA library with cDNA derived from mRNA prepared after infection of potato leaves with P. infestans and from mRNA prepared after growth of P. infestans on a basic medium in culture. Amongst the up-regulated P. infestans sequences were the ipiO and ipiB genes, each of which is a member of a cluster of related sequences [63]. ...
... [63] Judelson et al. [55] isolated a number of in planta induced (ipi ) genes by differential screening of a P. infestans genomic DNA library with cDNA derived from mRNA prepared after infection of potato leaves with P. infestans and from mRNA prepared after growth of P. infestans on a basic medium in culture. Amongst the up-regulated P. infestans sequences were the ipiO and ipiB genes, each of which is a member of a cluster of related sequences [63]. ...
... Fungicides can slow or stop the development of new symptoms if applied in a timely fashion, but fungicides will not cure existing light blight symptoms [71]. In Ethiopia the first spray with Ridomil MZ 63.5% WP at a rate of 2 kg ha -1 followed by 2-3 sprays (need base application) of Dithane M-45 (Mancozeb) at a rate of 3 kg ha -1 were found to be effective in controlling late blight [72,73]. He also reported that, reduced rates of Ridomil application resulted in better management of potato late blight with the highest marginal rate of return. ...
Potato is the world’s number one non-grain food commodity, with production reaching 325 million tons. Some
inherent qualities give potato a competitive edge over the leading food crops. It is able to produce more protein and
carbohydrates per unit area than cereals. Among all the crops grown worldwide, is known to suffer the greatest losses
from disease attack. Among those diseases late blight and bacterial wilt are the most economical diseases which
incurred 100% yield loss. The main Objective of this review was to understand very well the recent advances on
potato late blight disease management up to gene for gene level and incorporate these recent molecular advances
with the conventional one for mitigating the virulence spectrum of the pathogen. The population of P. infestans
characterized using molecular markers has led to better understanding of pathogen at molecular level.
Mitochondrial DNA haplotyping of P. infestans has revealed that mt Ia is displacing the other haplotypes
globally at a faster rate.
... We hypothesised that the high-affinity RGD-binding sites at the plasma membrane may be the plant counterpart of integrins, and to further identify these membrane components we developed a photoaffinity method to cross-link an RGD-containing peptide to its potential targets on purified plasma membrane. We identified an 80-kDa RGDbinding protein that is strongly selective towards the RGD sequence and show that this 80-kDa plasma membrane protein functions as a receptor for the RGD-containing protein IPI-O from the oomycete plant pathogen Phytophthora infestans [19]. Finally, we show that the IPI-O protein promotes the disruption of cell wall-membrane contacts in A. thaliana. ...
... RGD motif receptor in Arabidopsis thaliana quence [19], was cloned into the expression vector pMALc [maltose binding protein (MBP) vectors; New England Biolabs] resulting in pPIN18c. Within the ipiO1 ORF, two individual mutations in the RGD tripeptide motif were obtained using PCR-mediated mutagenesis and Amplitaq polymerase (Perkin Elmer) with pPIN18c as template. ...
... The RGD motif in the P. infestans IPI-O protein is exposed to the surface The deduced amino acid sequence of the P. infestans ipiO gene [19] revealed the presence of an arginine-glycineaspartic acid (RGD) tripeptide motif located at positions 54 -56, a putative N-terminal signal sequence (positions 1-21) suggesting that the IPI-O protein is secreted, and a potential N-glycosylation site (NTSD) at positions 66 -69 ( fig. 1 A). An HCA plot was drawn in order to predict the structural features of the IPI-O protein and, in particular, to evaluate the accessibility of the RGD sequence as a potential recognition motif ( fig. 1 B). ...
The RGD tripeptide sequence, a cell adhesion motif present in several extracellular matrix proteins of mammalians, is involved in numerous plant processes. In plant-pathogen interactions, the RGD motif is believed to reduce plant defence responses by disrupting adhesions between the cell wall and plasma membrane. Photoaffin- ity cross-linking of (125I)-azido-RGD heptapeptide in the presence of purified plasma membrane vesicles of Ara- bidopsis thaliana led to label incorporation into a single protein with an apparent molecular mass of 80 kDa. In- corporation could be prevented by excess RGD peptides, but also by the IPI-O protein, an RGD-containing protein secreted by the oomycete plant pathogen Phytophthora
... In contrast to other eukaryotes, oomycetes seem to lack canonical TATA-box elements [22]. However, many genes have an Inr-element that resembles the general eukaryotic Inr-element [23,24]; an element that is sufficient to direct the accurate transcription in the absence of other elements242526. Interestingly, oomycetes have a flanking promoter region (FPR) downstream of the Inr-element that has not yet been described as an important functional region in other eukaryotes [24]. ...
... To validate our method, we first surveyed the 19 obtained motifs for similarity to known eukaryotic promoter elements. Pregenome analyses of the upstream regions of a small set of oomycete genes have identified a Inr/FPR-element as a core promoter element [23,24]. Indeed, our in silico approach recovered the previously described oomycete-specific Inr/FPR element (motif-0). ...
... Hence, in combination with a stringent significance cutoff, the biased motifs would not be able to identify all occurrences in the genome and consequently underestimate the true abundance. This is indeed the case for the upstream region of ipiO1 gene in which the Inr/FPR-element was initially described [23]. If we specifically searched for the occurrence of this motif in the upstream region, we could identify its occurrence at 28 nt upstream of the TLS. ...
Plant infection by oomycete pathogens is a complex process. It requires precise expression of a plethora of genes in the pathogen that contribute to a successful interaction with the host. Whereas much effort has been made to uncover the molecular systems underlying this infection process, mechanisms of transcriptional regulation of the genes involved remain largely unknown. We performed the first systematic de-novo DNA motif discovery analysis in Phytophthora. To this end, we utilized the genome sequence of the late blight pathogen Phytophthora infestans and two related Phytophthora species (P. ramorum and P. sojae), as well as genome-wide in planta gene expression data to systematically predict 19 conserved DNA motifs. This catalog describes common eukaryotic promoter elements whose functionality is supported by the presence of orthologs of known general transcription factors. Together with strong functional enrichment of the common promoter elements towards effector genes involved in pathogenicity, we obtained a new and expanded picture of the promoter structure in P. infestans. More intriguingly, we identified specific DNA motifs that are either highly abundant or whose presence is significantly correlated with gene expression levels during infection. Several of these motifs are observed upstream of genes encoding transporters, RXLR effectors, but also transcriptional regulators. Motifs that are observed upstream of known pathogenicity-related genes are potentially important binding sites for transcription factors. Our analyses add substantial knowledge to the as of yet virtually unexplored question regarding general and specific gene regulation in this important class of pathogens. We propose hypotheses on the effects of cis-regulatory motifs on the gene regulation of pathogenicity-related genes and pinpoint motifs that are prime targets for further experimental validation.
... In the early 1990s, a number of in planta induced (ipi) genes were isolated from P. infestans by differential screening of a P. infestans genomic DNA library with cDNA prepared from infected potato and cDNA prepared from mycelium grown in basic culture medium (Pieterse et al. 1991(Pieterse et al. , 1993(Pieterse et al. , 1994a(Pieterse et al. , 1994b. These genes included the ipiB and ipiO gene families, each of which is clustered in the genome (Pieterse et al. 1994a(Pieterse et al. , 1994b). ...
... In the early 1990s, a number of in planta induced (ipi) genes were isolated from P. infestans by differential screening of a P. infestans genomic DNA library with cDNA prepared from infected potato and cDNA prepared from mycelium grown in basic culture medium (Pieterse et al. 1991(Pieterse et al. , 1993(Pieterse et al. , 1994a(Pieterse et al. , 1994b. These genes included the ipiB and ipiO gene families, each of which is clustered in the genome (Pieterse et al. 1994a(Pieterse et al. , 1994b). An ipiO gene has since been shown to be up-regulated in invading hyphae during the early stages of infection (van West et al. 1998). ...
Late blight, caused by the oomycete Phytophthora infestans, is one of the most serious constraints to potato
production worldwide. An understanding of the basic mechanisms of infection by the pathogen and of effective defence by the host, are required for informed strategies seeking either targeted chemical control of the pathogen or the development of durable resistance in the plant. An initial insight into the molecular events underpinning P. infestans –potato interactions can be gained by determining the interaction transcriptome, the sum of the transcripts from both host and pathogen that are produced during their association. To this end, projects with expressed sequence tags are ongoing and provide an overview of the genes expressed in particular growth conditions, cell types, and developmental stages, in both potato and P. infestans, and allow significant sampling of genes expressed as part of the interaction transcriptome. As many genes required for pathogenicity or resistance are transcriptionally induced, methods to enrich for up-regulated genes, such as the powerful technique of suppression subtractive hybridization based on polymerase chain reaction, are also being used to identify candidate genes required in P. infestans for infection of the host or in potato for defence against the pathogen.
... PexRD6 was the first cloned RxLR effector candidate (Pieterse, Vanwest et al. 1994) and represents the ipiO family (ipi, in planta induced). This is a highly diverse family and many of its variants were included in this profiling study. ...
... The first published GRPs from P. infestans, i.e. IPI-B, were already identified in a time that P. infestans was about to enter the genomics era (Pieterse, Vanwest et al. 1994). The theme that zoospores should adhere to their host during the zoosporic stage could indicate (e.g. ...
Late Blight (LB) is a major threat to potato production worldwide and caused by the pathogen Phytophthora infestans. Using sophisticated equipment, P. infestans initially enters the potato cell where it is able to secrete specialized proteins aimed to silence the plant primary immune system, using so-called haustoria. Many of such proteins belong to the class of RxLR effectors, of which approximately 500 are predicted in the P. infestans genome. Several of these proteins are intercepted by NBS-LRR (R-)genes with finally results in an hypersensitive cell death response , thus initiating a secondary resistance layer. In this thesis we searched for major resistance genes in wild potato species. Through transiently expressing RxLR genes in potato plants with novel resistance profiles, leads to unknown R-Avr combinations were scouted. In a second phase, such candidate Avr genes were followed in potato populations segregating for the resistances. Finally, a total of eleven new avirulence candidates were revealed and from these, three were confirmed using additional assays. Among the studied potato genotypes was the LB-resistant potato cultivar Sarpo Mira which was shown to contains at least five R-genes. Interestingly, QTLs for LB resistance can be precisely mapped using an effector approach, which opens possibilities for cloning such genes.
... 179 Similarly, in plants, synthetic peptides and proteins containing the RGD motif disrupt the adhesion between plasma membrane and cell wall [180][181][182] which leads to altered physiological processes affecting plant development, [183][184][185][186] gravity sensing, 187 and the interaction between plants and microbes. [188][189][190][191][192][193][194] Also, the RGDBP and VPS9 effector proteins from Puccinia graminis are located in the apoplast of the plant cell 35 and initiate signaling events which lead to Rpg1mediated stem rust resistance. 195 Shenchou et al. identified an 80-kDa plasma membrane protein as receptor for the RGD-containing protein IPI-O from P. infestans, which promotes the disruption of the cell wall-membrane contacts in Arabidopsis. ...
Effector proteins play important roles in the infection by pathogenic oomycetes and fungi or the colonization by endophytic and mycorrhizal fungi. They are either translocated into the host plant cells via specific translocation mechanisms and function in the host's cytoplasm or nucleus, or they reside in the apoplast of the plant cells and act at the extracellular host-microbe interface. Many effector proteins possess conserved motifs (such as the RXLR, CRN, LysM, RGD, DELD, EAR, RYWT, Y/F/WXC or CFEM motifs) localized in their N- or C-terminal regions. Analysis of the functions of effector proteins, especially so-called "core effectors", is crucial for the understanding of pathogenicity/symbiosis mechanisms and plant defense strategies, and helps to develop breeding strategies for pathogen-resistant cultivars, and to increase crop yield and quality as well as abiotic stress resistance. This review summarizes current knowledge about these effector proteins with the conversed motifs and their involvement in pathogenic or mutualistic plant/fungal interactions.
... There is also an IpiB3-like protein (PITG_11341). IpiB proteins are glycine-rich proteins that were already discovered in 1994, in a screen for genes upregulated in the early stages of the P. infestans-potato interaction, so during or shortly after infection (39,40). As yet, the function of IpiB proteins is not known, but based on their glycine-rich nature it is tempting to speculate that they are structural proteins that play a role in establishing infection structures during the early stages of infection. ...
The oomycete Phytophthora infestans is the most harmful pathogen of potato. It causes the disease late blight, which generates increased yearly costs of up to one billion euro in the EU alone and is difficult to control. We have performed a large-scale quantitative proteomics study of six P. infestans life stages with the aim to identify proteins that change in abundance during development, with a focus on pre-infectious life stages. Over 10 000 peptides from 2061 proteins were analysed. We identified a number of abundance profiles of proteins that were up- or downregulated in different combinations of life stages. One of these profiles contained 59 proteins that were more abundant in germinated cysts and appressoria. A large majority of these proteins were not previously recognized as being appressorial proteins or involved in the infection process. Among those are proteins with putative roles in transport, amino acid metabolism, pathogenicity (including one RXLR effector) and cell wall structure modification. We analysed the expression of the genes encoding nine of these proteins using RT-qPCR and found an increase in transcript levels during disease progression, in agreement with the hypothesis that these proteins are important in early infection. Among the nine proteins was a group involved in cell wall structure modification and adhesion, including three closely related, uncharacterized proteins encoded by PITG_01131, PITG_01132, and PITG_16135, here denoted Piacwp1-3 Transient silencing of these genes resulted in reduced severity of infection, indicating that these proteins are important for pathogenicity. Our results contribute to further insight into P. infestans biology, and indicate processes that might be relevant for the pathogen while preparing for host cell penetration and during infection. The mass spectrometry data have been deposited to ProteomeXchange via the PRIDE partner repository with the dataset identifier PXD002446.
... This is increasingly difficult with the number of R genes that are introduced, as late blight resistance assays can often not clearly measure the functional expression of all introduced R genes. We found that the response of Rpi-sto1, a close relative of RB, to its cognate Avr (IPI-O; Pieterse et al. 1994;Vleeshouwers et al. 2008) is strictly correlated with the level of resistance in transgenic plants (Zhu et al. 2013). Thus, Avr responsiveness is the most preferred tool to validate functional expression of R genes in stacks, both in classical and GM breeding. ...
Key message:
The potato late blight resistance gene R8 has been cloned. R8 is found in five late blight resistant varieties deployed in three different continents. R8 recognises Avr8 and is homologous to the NB-LRR protein Sw-5 from tomato. The broad spectrum late blight resistance gene R8 from Solanum demissum was cloned based on a previously published coarse map position on the lower arm of chromosome IX. Fine mapping in a recombinant population and bacterial artificial chromosome (BAC) library screening resulted in a BAC contig spanning 170 kb of the R8 haplotype. Sequencing revealed a cluster of at least ten R gene analogues (RGAs). The seven RGAs in the genetic window were subcloned for complementation analysis. Only one RGA provided late blight resistance and caused recognition of Avr8. From these results, it was concluded that the newly cloned resistance gene was indeed R8. R8 encodes a typical intracellular immune receptor with an N-terminal coiled coil, a central nucleotide binding site and 13 C-terminal leucine rich repeats. Phylogenetic analysis of a set of representative Solanaceae R proteins shows that R8 resides in a clearly distinct clade together with the Sw-5 tospovirus R protein from tomato. It was found that the R8 gene is present in late blight resistant potato varieties from Europe (Sarpo Mira), USA (Jacqueline Lee, Missaukee) and China (PB-06, S-60). Indeed, when tested under field conditions, R8 transgenic potato plants showed broad spectrum resistance to the current late blight population in the Netherlands, similar to Sarpo Mira.
... Avr-blb1 turned out to be identical to the in planta induced gene ipiO, which was initially postulated to be involved www.annualreviews.org • Resistance to Late Blight in Potato in pathogenicity based on its expression profile (102,103,141) (Table 2, Figure 3). In the genome of particular P. infestans strains, ipiO is present in two copies in inverted orientation (41, 103). ...
Potato (Solanum tuberosum) is the world’s third-largest food crop. It
severely suffers from late blight, a devastating disease caused by Phytophthora
infestans. This oomycete pathogen secretes host-translocated
RXLR effectors that include avirulence (AVR) proteins, which are
targeted by resistance (R) proteins from wild Solanum species. Most
Solanum Rgenes appear to have coevolved with P. infestans at its center of
origin in central Mexico. Various R and Avr genes were recently cloned,
and here we catalog characterized R-AVR pairs.We describe themechanisms
that P. infestans employs for evading R protein recognition and
discuss partial resistance and partial virulence phenotypes in the context
of our knowledge of effector diversity and activity. Genome-wide
catalogs of P. infestans effectors are available, enabling effectoromics
approaches that accelerate R gene cloning and specificity profiling. Engineering
R genes with expanded pathogen recognition has also become
possible. Importantly, monitoring effector allelic diversity in pathogen
populations can assist in R gene deployment in agriculture.
... Genome sequencing confirmed that the ORF is present in the genome and revealed an intron of 55 nt long, ranging from 74 nt up to 129 nt. Also, the oomycete conserved sequence motif in the promoter region of SpHtp1 was identified 35 nt upstream of the start codon (Pieterse et al., 1994;McCleod et al., 2004) (Fig. S2). BLASTP analysis of SpHtp1 yielded sequence similarity only to regions of low complexity in other proteins such as the elicitin 6 precursor proteins of Phytophthora medicaginis, Phytophthora ramorum and Phytophthora sojae (E-values 9e-19, 2e-16, 3e-16 and 52%, 68% and 51% identity, respectively). ...
The fish pathogenic oomycete Saprolegnia parasitica causes the disease Saprolegniosis in salmonids and other freshwater fish, resulting in considerable economic losses in aquaculture. Very little is known about the molecular and cellular mechanisms underlying the infection process of fish pathogenic oomycetes. In order to investigate the interaction in detail, an in vitro infection assay using an Oncorhynchus mykiss (rainbow trout) cell line (RTG-2) was developed. In a zoospore/cyst cDNA library, we identified the ORF SpHtp1, which encodes a secreted protein containing an RxLR motif. Detailed expression analysis indicated that SpHtp1 is highly expressed in zoospores/cysts from S. parasitica and in the very early stages of infection on RTG-2 cells, when compared with in vitro-grown mycelium. Moreover, the protein, SpHtp1, was found to translocate into the RTG-2 trout cells, during the interaction with S. parasitica, and also when the RTG-2 cells were treated with recombinant SpHtp1 fused to a C-terminal His-tag. These findings suggest that protein translocation could play an important role in Saprolegniosis.
... Class I and II ipiO proteins, members of the RxLR family of putative effectors, induce avirulence responses in potato lines containing RB [20][21][22]. After inoculation (24, 48 hpi), class I ipiO transcripts were detected (though in low abundance) in five of six tuber samples collected from the P. infestans-inoculated WT line but from no samples derived from the +RB line (Additional file 10). ...
Background
The late blight pathogen Phytophthora infestans can attack both potato foliage and tubers. Although interaction transcriptome dynamics between potato foliage and various pathogens have been reported, no transcriptome study has focused specifically upon how potato tubers respond to pathogen infection. When inoculated with P. infestans, tubers of nontransformed ‘Russet Burbank’ (WT) potato develop late blight disease while those of transgenic ‘Russet Burbank’ line SP2211 (+RB), which expresses the potato late blight resistance gene RB (Rpi-blb1), do not. We compared transcriptome responses to P. infestans inoculation in tubers of these two lines.
Results
We demonstrated the practicality of RNA-seq to study tetraploid potato and present the first RNA-seq study of potato tuber diseases. A total of 483 million paired end Illumina RNA-seq reads were generated, representing the transcription of around 30,000 potato genes. Differentially expressed genes, gene groups and ontology bins that exhibited differences between the WT and +RB lines were identified. P. infestans transcripts, including those of known effectors, were also identified.
Conclusion
Faster and stronger activation of defense related genes, gene groups and ontology bins correlate with successful tuber resistance against P. infestans. Our results suggest that the hypersensitive response is likely a general form of resistance against the hemibiotrophic P. infestans—even in potato tubers, organs that develop below ground.
... oomycetes have diploid nuclei, whereas fungi have haploid nuclei; oomycetes use diaminopimelate (or DAP) pathway for lysine synthesis (Barr, 1983), while fungi use the aminoadipate (or AAA) pathway (Vogel, 1965); promoters from plants, fungi, and animals have no detectable activity in oomycetes (Judelson, Tyler, & Michelmore, 1992); most cloned genes in oomycetes lack the TATA element in their promoter regions; and instead, a 19-nt core sequence (McLeod, Smart, & Fry, 2004;Pieterse et al., 1994) similar to the initiator element (Quon, Delgadillo, Khachi, Smale, & Johnson, 1994) was overrepresented. Based on modern molecular and biochemical analyses, oomycetes are more closely related to brown algae and diatoms and fall in the kingdom Stramenopila ( Fig. 12.1) (Baldauf et al., 2000;Cavalier-Smith, 2000; The tree is adapted from that of Baldauf, Roger, Wenk-Siefert, and Doolittle (2000) that is based on a concatenation of six highly conserved proteins. ...
Phytophthora sp. is a genus in the oomycetes, which are similar to filamentous fungi in morphology and habitat, but phylogenetically more closely related to brown algae and diatoms and fall in the kingdom Stramenopila. In the past few years, several viruses have been characterized in Phytophthora species, including four viruses from Phytophthora infestans, the late blight pathogen, and an endornavirus from an unnamed Phytophthora species from Douglas fir. Studies on Phytophthora viruses have revealed several interesting systems. Phytophthora infestans RNA virus 1 (PiRV-1) and PiRV-2 are likely the first members of two new virus families; studies on PiRV-3 support the establishment of a new virus genus that is not affiliated with established virus families; PiRV-4 is a member of Narnaviridae, most likely in the genus Narnavirus; and Phytophthora endornavirus 1 (PEV1) was the first nonplant endornavirus at the time of reporting. Viral capsids have not been found in any of the above-mentioned viruses. PiRV-1 demonstrated a unique genome organization that requires further examination, and PiRV-2 may have played a role in late blight resurgence in 1980s-1990s.
... Plant, animal, and fungal promoters were shown to work poorly in the potato late blight pathogen Phytophthora infestans, which suggested divergence of the transcriptional apparatus of oomycetes [11]. Researchers examining the few genes that were available prior to the development of whole-genome sequences reported that nearly all oomycete promoters contain a 16 to 19 nt region near the TSS, which contained a 7 nt INR-like element at its 5 0 end followed by an approximately 9 nt sequence named Flanking Promoter Region or FPR [12,13]. Some genes were reported to contain TATA-like motifs, but these were not functionally tested and could be spurious matches [14,15]. ...
Background
The core promoter is the region flanking the transcription start site (TSS) that directs formation of the pre-initiation complex. Core promoters have been studied intensively in mammals and yeast, but not in more diverse eukaryotes. Here we investigate core promoters in oomycetes, a group within the Stramenopile kingdom that includes important plant and animal pathogens. Prior studies of a small collection of genes proposed that oomycete core promoters contain a 16 to 19 nt motif bearing an Initiator-like sequence (INR) flanked by a novel sequence named FPR, but this has not been extended to whole-genome analysis.
Results
We used expectation maximization to find over-represented motifs near TSSs of Phytophthora infestans, the potato blight pathogen. The motifs corresponded to INR, FPR, and a new element found about 25 nt downstream of the TSS called DPEP. TATA boxes were not detected. Assays of DPEP function by mutagenesis were consistent with its role as a core motif. Genome-wide searches found a well-conserved combined INR+FPR in only about 13% of genes after correcting for false discovery, which contradicted prior reports that INR and FPR are found together in most genes. INR or FPR were found alone near TSSs in 18% and 7% of genes, respectively. Promoters lacking the motifs had pyrimidine-rich regions near the TSS. The combined INR+FPR motif was linked to higher than average mRNA levels, developmentally-regulated transcription, and functions related to plant infection, while DPEP and FPR were over-represented in constitutively-expressed genes. The INR, FPR, and combined INR+FPR motifs were detected in other oomycetes including Hyaloperonospora arabidopsidis, Phytophthora sojae, Pythium ultimum, and Saprolegnia parasitica, while DPEP was found in all but S. parasitica. Only INR seemed present in a non-oomycete stramenopile.
Conclusions
The absence of a TATA box and presence of novel motifs show that the oomycete core promoter is diverged from that of model systems, and likely explains the lack of activity of non-oomycete promoters in Phytophthora transformants. The association of the INR+FPR motif with developmentally-regulated genes shows that oomycete core elements influence stage-specific transcription in addition to regulating formation of the pre-initiation complex.
... st, potato, and in non-host interactions (see below) with a variety of Nicotiana spp. Many of the genes with a key role in either host defence or pathogenicity will be up-regulated during the P. infestans –plant association. A number of methods exist for isolating such differentially expressed genes. Pieterse et al . (1991 Pieterse et al . ( , 1993 Pieterse et al . ( , 1994a,b) isolated a number of in planta induced ( ipi ) genes by differential screening of a P. infestans genomic DNA library with cDNA derived from mRNA prepared after infection of potato leaves with P. infestans and from mRNA prepared after growth of P. infestans on a basic medium in culture. Amongst the up-regulated P. infestans sequences ...
Unlabelled:
summary Phytophthora infestans, cause of late-blight, is the most devastating disease of potato world-wide. Recent years have seen a dramatic intensification in molecular biological studies of P. infestans, including the development of novel tools for transformation and gene silencing and the resources for genetical, transcriptional and physical mapping of the genome. This review will focus on the increasing efforts to use these resources to discover the genetic bases of pathogenicity, avirulence and host-specificity.
Taxonomy:
Phytophthora infestans (Mont.) de Bary-Kingdom Chromista, Phylum Oomycota, Order Peronosporales, Family Peronosporaceae, Genus Phytophthora, of which it is the type species.
Host range:
Infects a wide range of solanaceous species. Economically important hosts are potato, tomato, eggplant and some other South American hosts (tree tomato and pear melon) on which it causes late blight. Disease symptoms: Infected foliage is initially yellow, becomes water soaked and eventually blackens. Leaf symptoms comprise purple-black or brown-black lesions at the leaf tip, later spreading across the leaf to the stem. Whitish masses of sporangia develop on the underside of the leaf. Tubers become infected later in the season and, in the early stages, consist of slightly brown or purple blotches on the skin. In damp soils the tuber decays rapidly before harvest. Tuber infection is quickly followed by secondary fungal or bacterial infection known as 'wet rot'. Useful web sites:http://www.ncgr.org/pgc/; http://www.oardc.ohio-state.edu/phytophthora/.
... from the nightshade Solanum bulbocastanum (Champouret et al., 2009). In the pre-genomic era comparative analysis that could have led to the discovery of the RXLR–dEER domain in IPI-O was not yet feasible; at that time the only potentially interesting feature that could be distinguished in IPI-O was the presence of a cell attachment motif (Pieterse et al., 1994 ). This motif consisting of the tripeptide sequence Arg-Gly-Asp (RGD) partly overlaps with the RXLR motif in IPI-O, i.e., RXLRGD. ...
Plants are constantly beset by pathogenic organisms. To successfully infect their hosts, plant pathogens secrete effector proteins, many of which are translocated to the inside of the host cell where they manipulate normal physiological processes and undermine host defense. The way by which effectors cross the frontier to reach the inside of the host cell varies among different classes of pathogens. For oomycete plant pathogens – like the potato late blight pathogen Phytophthora infestans – it has been shown that effector translocation to the host cell cytoplasm is dependent on conserved amino acid motifs that are present in the N-terminal part of effector proteins. One of these motifs, known as the RXLR motif, has a strong resemblance with a host translocation motif found in effectors secreted by Plasmodium species. These malaria parasites, that reside inside specialized vacuoles in red blood cells, make use of a specific protein translocation complex to export effectors from the vacuole into the red blood cell. Whether or not also oomycete RXLR effectors require a translocation complex to cross the frontier is still under investigation. For one P. infestans RXLR effector named IPI-O we have found a potential host target that could play a role in establishing the first contact between this effector and the host cell. This membrane spanning lectin receptor kinase, LecRK-I.9, interacts with IPI-O via the tripeptide RGD that overlaps with the RXLR motif. In animals, RGD is a well-known cell adhesion motif; it binds to integrins, which are membrane receptors that regulate many cellular processes and which can be hijacked by pathogens for either effector translocation or pathogen entry into host cells.
... A total of 1002 sequences corresponding to the 5 end of the cDNA inserts were generated using primer M13 reverse. In Phytophthora transcripts, the 5 untranslated regions are typically short, ranging from about 50 to 70 nucleotides (Pieterse et al., 1994). Therefore, the DNA sequences obtained with primer M13 reverse are highly informative and generally overlap an open reading frame even when the cDNA is full length. ...
A total of 1000 expressed sequence tags (ESTs) corresponding to 760 unique sequence sets were identified using random sequencing of clones from a cDNA library constructed from mycelial RNA of Phytophthora infestans. A number of software programs, represented by a relational database and an analysis pipeline, were developed for the automated analysis and storage of the EST sequence data. A set of 419 nonredundant sequences, which correspond to a total of 632 ESTs (63.2%), were identified as showing significant matches to sequences deposited in public databases. A putative cellular identity and role was assigned to all 419 sequences. All major functional categories were represented by at least several ESTs. Four novel cDNAs containing sequences related to elicitins, a family of structurally related proteins that induce the hypersensitive response and condition avirulence of P. infestans on Nicotiana plants, were among the most notable genes identified. Two of these elicitin-like cDNAs were among the most abundant cDNAs examined. The set also contained several ESTs with high sequence similarity to unique plant genes.
Filamentous (oomycete and fungal) plant pathogens deliver cytoplasmic effector proteins into host cells to facilitate disease. How RXLR effectors from the potato late blight pathogen Phytophthora infestans enter host cells is unknown. One possible route involves clathrin-mediated endocytosis (CME). Transient silencing of NbCHC, encoding clathrin heavy chain, or the endosome marker gene NbAra6 encoding a Rab GTPase in the model host Nicotiana benthamiana, attenuated P. infestans infection and reduced translocation of RXLR effector fusions from transgenic pathogen strains into host cells. By contrast, silencing PP1c isoforms, susceptibility factors not required for endocytosis, reduced infection but did not attenuate RXLR effector uptake. Endosome enrichment by ultracentrifugation and sucrose gradient fractionation revealed co-localization of RXLR effector Pi04314-RFP with clathrin-coated vesicles. Immunopurification of clathrin- and NbAra6-associated vesicles during infection showed that RXLR effectors Pi04314-RFP and AvrBlb1-RFP, but not apoplastic effector PiSCR74-RFP, were co-immunoprecipitated during infection with pathogen strains secreting these effectors. Tandem mass spectrometry analyses of proteins co-immunoprecipitated with NbAra6-GFP during infection revealed enrichment of host proteins associated with endocytic vesicles alongside multiple pathogen RXLR effectors, but not apoplastic effectors, including PiSCR74, which do not enter host cells. Our data show that uptake of P. infestans RXLR effectors into plant cells occurs via CME.
The sections in this article are
Introduction
History of Late Blight
Economic and Social Impact of P hytophthora Plant Pathogens
P hytophthora Infestans and its Taxonomic Position
The Disease Cycle of P hytophthora Infestans
The Plant Response
Future Perspectives
Acknowledgements
The oomycete potato blight pathogen Phytophthora infestans secretes a diverse set of proteins to manipulate host plant immunity. However, there is limited knowledge about how and where they are secreted during infection. Here we used the endoplasmic reticulum (ER)-to-Golgi secretion pathway inhibitor brefeldin A (BFA) in combination with liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) to identify extracellular proteins from P. infestans that were conventionally secreted from in vitro-cultured hyphae. We identified 19 proteins with predicted signal peptides that potentially influence plant interactions for which secretion was attenuated by BFA. In addition to inhibition by the apoplastic effector EPIC1, a cysteine protease inhibitor, we show that secretion of the cell wall-degrading pectinesterase enzyme PE1 and the microbe-associated molecular pattern (MAMP)-like elicitin INF4 was inhibited by BFA in vitro and in planta, demonstrating that these proteins are secreted by the conventional, Golgi-mediated pathway. For comparison, secretion of a cytoplasmic RXLR (Arg-[any amino acid]-Leu-Arg) effector, Pi22926, was not inhibited by BFA. During infection, whereas INF4 accumulated outside the plant cell, RXLR effector Pi22926 entered the plant cell and accumulated in the nucleus. The P. infestans effectors, the PE1 enzyme, and INF4 were all secreted from haustoria, pathogen structures that penetrate the plant cell wall to form an intimate interaction with the host plasma membrane. Our findings show the haustorium to be a major site of both conventional and nonconventional secretion of proteins with diverse functions during infection.
Late blight of potato, caused by the oomycete pathogen Phytophthora infestans, is persistent and costly disease of this important crop plant. Breeders have continually struggled with the evolution and introduction of new pathogen genotypes that threaten to overcome major resistance genes incorporated into new varieties. Wild species relatives of potato have been a valuable resource for the identification of novel resistance genes including the RB gene, from the diploid wild potato species Solanum bulbocastanum. The RB gene confers partial resistance to most P. infestans genotypes through its recognition of members of the corresponding pathogen effector protein family IPI-O. Multiple alleles are present at the IPI-O locus and while some alleles are recognized by RB to elicit host resistance (e.g. IPI-O1, IPI-O2), others are able to elude detection (e.g. IPI-O4). In our previous research, we found that in planta expression of P. infestans effector IPI-O4 is able to suppress the HR elicited by IPI-O1 in the presence of RB. Additionally, we have observed that P. infestans lineages containing IPI-O4 are able to cause more disease on RB plants compared to those without IPI-O4. This led to a hypothesis that the presence of IPI-O4 results in suppression of RB. In this study, we provide evidence that in planta over-expression of IPI-O4 is able to suppress RB-mediated resistance, causing enlarged lesions in RB containing K41 potato lines. The results of this study indicate that even subtle effects from host or pathogen factors during the early stages of infection can heavily influence the ultimate outcome of the interaction.
Potato late blight, caused by the oomycete pathogen Phytophthora infestans, is one of the most destructive plant diseases. Despite decades of intensive breeding efforts, it remains a threat to potato production worldwide, because newly evolved pathogen strains have overcome major resistance genes quickly. The RB protein, from the diploid wild potato species Solanum bulbocastanum, confers partial resistance to most P. infestans strains through its recognition of members of the corresponding pathogen effector protein family IPI-O. IPI-O comprises a multigene family and while some variants are recognized by RB to elicit host resistance (e.g., IPI-O1 and IPI-O2), others are able to elude detection (e.g., IPI-O4). IPI-O1 is almost ubiquitous in global P. infestans strains while IPI-O4 is more rare. No direct experimental evidence has been shown to demonstrate the effect of IPI-O on pathogen virulence in the P. infestans-potato pathosystem. Here, our work has demonstrated that in planta expression of both IPI-O1 and IPI-O4 increases P. infestans aggressiveness resulting in enlarged lesions in potato leaflets. We have previously shown that IPI-O4 has gained the ability to suppress the hypersensitive response induced by IPI-O1 in the presence of RB. In this study, our work has shown that this gain-of-function of IPI-O4 does not compromise its virulence effect, as IPI-O4 overexpression results in larger lesions than IPI-O1. We have also found that higher expression of IPI-O effectors correlates with enlarged lesions, indicating that IPI-O can contribute to virulence quantitatively. In summary, this study has provided accurate and valuable information on IPI-O's virulence effect on the potato host.
Pathogenic fungi have diverse growth lifestyles that support fungal colonization on plants. Successful colonization and infection for all lifestyles depends upon the ability to modify living host plants to sequester the necessary nutrients required for growth and reproduction. Secretion of virulence determinants referred to as ‘effectors’ is assumed to be the key governing factor that determines host infection and colonization. Effector proteins are capable of suppressing plant defense responses and alter plant physiology to accommodate fungal invaders. This review focuses on effector molecules of biotrophic and hemibiotrophic plant pathogenic fungi, and the mechanism required for the release and uptake of effector molecules by the fungi and plant cells, respectively. We also place emphasis on the discovery of effectors, difficulties associated with predicting the effector repertoire, and fungal genomic features that have helped promote effector diversity leading to fungal evolution. We discuss the role of specific effectors found in biotrophic and hemibiotrophic fungi and examine how CRISPR technology may provide a new avenue for accelerating our ability in the discovery of fungal effector function.
In this study we investigated the genetic control of avirulence in the diploid oomycete pathogen Phytophthora infestans, the causal agent of late blight on potato. The dominant avirulence (Avr) genes matched six race-specific resistance genes introgressed in potato from a wild Solanum species. AFLP markers linked to Avr genes were selected by bulked segregant analysis and used to construct two high-density linkage maps, one containing Avr4 (located on linkage group A2-a) and the other containing a cluster of three tightly linked genes, Avr3, Avr10, and Avr11 (located on linkage group VIII). Bulked segregant analysis also resulted in a marker linked to Avr1 and this allowed positioning of Avr1 on linkage group IV. No bulked segregant analysis was performed for Avr2, but linkage to a set of random markers placed Avr2 on linkage group VI. Of the six Avr genes, five were located on the most distal part of the linkage group, possibly close to the telomere. The high-density mapping was initiated to facilitate future positional cloning of P. infestans Avr genes.
RXLR effector candidates used in this study. PexRD, PexRD family members (nr), Agrobacterium tumefaciens clones, known genes, the Phytopthora infestans strain and amino acid sequences are presented. PexRD and A. tumefaciens clones correspond to Table 2.
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Of the numerous pest and diseases that affect potato growth and tuber production, diseases caused by three oomycete species pathogens, such as late blight, pink rot and watery wound rot (leak), often result in the greatest crop-production and post-harvest losses. Specifically, potato late blight can completely destroy a growing, healthy crop in a matter of a couple of weeks under favourable climatic conditions. Meanwhile, potato pink rot and leak (watery wound rot) can result in major or complete post-harvest tuber losses even when crop growth has visibly been healthy. Extensive knowledge about disease occurrence, impact and symptoms has been acquired in the past 50 years about these diseases. Meanwhile, improved investigation techniques have begun to reveal complexities regarding aspects of pathogen biology and disease development that were poorly understood. Similarly, advances in agricultural production systems have led to evergreater needs for improved disease management methods. However, the increasing demands for high quality, low priced and safe food produced in an environmentally benign manner places further needs for more research on interactions among the pathogen - host - environment - human complex. This chapter summarizes various aspects of these issues and contains selected references for further information on these disease issues.
Data on molecular mapping of chromosomal heteromorphism S1 locus of plant pathogenic fungus Phytophthora infestans are presented. The F1 progeny from two matings of field isolates was examined by means of PRC amplification with specific primers. Association of the given locus with the mating type gene in the linkage group III was confirmed. The possible role of the S1 locus in non-Mendelian inheritance of the mating type locus and abnormal behavior of the whole linkage group are discussed.
Phytophthora infestans is a destructive pathogen of potato and tomato, and a model system for oomycetes, a lineage of mostly filamentous eukaryotes related to diatoms and brown algae. P. infestans has had tremendous impacts on human history and is still a major threat to food security. Advances in our understanding of oomycete biology and evolution have resulted from analyses of the P. infestans genome, population studies, and tests of gene function enabled by technologies for transgene expression and silencing. P. infestans has a large repeat-rich genome with a complex organization of gene-dense and rapidly evolving gene-sparse regions. Transposable elements, horizontal gene transfer, novel gene fusions, and expansions of gene families encoding transporters, signaling proteins, and pathogenesis effectors have shaped the structure and function of the genome. Both classic transcription factor networks and epigenetics influence gene expression, which exhibits dynamic changes during development and infection. Insights into mechanisms of growth and dispersal, metabolism, pathogenesis, and defense against plant resistance and agrochemicals are leading to better strategies for managing P. infestans blights. This chapter describes these advances and challenges for future research in the post-genome era. © 2014 Springer-Verlag Berlin Heidelberg (outside the USA). All rights reserved.
Ever since the potato late blight epidemics of the mid-nineteenth century, members of the oomycete genus Phytophthora have emerged as major pathogens of innumerable crops. Nowadays, with more than fifty species recognized and with destructive diseases caused on thousands of plant species, Phytophthora remains an active subject of research and a nagging problem to farmers and growers. Renewed interest in Phytophthora diseases occurred in recent years with the reemergence of Phytophthora infestans, the Irish potato famine fungus. Late blight has turned into a global threat to potato and tomato production following a series of severe late blight epidemics that coincided with the migration to Europe and North America of aggressive A2 mating type strains (Fry and Goodwin, 1997a; Fry and Goodwin, 1997b). The International Potato Center estimated that worldwide losses in potato production caused by late blight have recently exceeded $3 billion annually, making P. infestans the single most important biotic constraint to global food production (Anonymous, 1996).
Members of the genus Phytophthora are destructive plant pathogens, causing significant economic losses worldwide. Extensive background knowledge exists on the transmission biology of the genus, but it is only in the last decade that the mechanisms underlying the success of the Phytophthoras as plant pathogens have begun to be elucidated at the cellular and molecular level. Using molecular genetic tools, substantial progress has been made in identifying key molecules involved in the pathogen infection cycle. Genomic approaches are now opening up new routes to gene discovery and promise to have a significant impact upon our understanding of the biology and pathology of this important group of organisms. This article reviews the tools and resources available for molecular genetic and genomic studies in Phytophthora and discusses the insights that these studies are giving into the molecular basis of the plant-pathogen relationship.
Many destructive diseases of plants and animals are caused by oomycetes, a group of eukaryotic pathogens important to agricultural, ornamental, and natural ecosystems. Understanding the mechanisms underlying oomycete virulence and the genomic processes by which those mechanisms rapidly evolve is essential to developing effective long-term control measures for oomycete diseases. Several common mechanisms underlying oomycete virulence, including protein toxins and cell-entering effectors, have emerged from comparing oomycetes with different genome characteristics, parasitic lifestyles, and host ranges. Oomycete genomes display a strongly bipartite organization in which conserved housekeeping genes are concentrated in syntenic gene-rich blocks, whereas virulence genes are dispersed into highly dynamic, repeat-rich regions. There is also evidence that key virulence genes have been acquired by horizontal transfer from other eukaryotic and prokaryotic species.
Introduction Avirulence Genes and RXLR-dEER-Family Effectors in Oomycete Plant Pathogens Potential Toxin Genes Hydrolases and Hydrolase Inhibitors Summary and Future Acknowledgments References
Recent advances in the classification of downy mildews and white blister rusts are presented from ordinal to species level. Using molecular data (mainly LSU of nuclear ribosomal DNA and ITS rDNA data, but also cox2, beta-tubulin and NADH genes), ordinal, family and generic circumscriptions have been reconsidered and changed during the last years; species circumscription and concepts are also changing. These rearrangements also lead to a reevaluation of the traditional morphological characters used for classification. The recent changes have various implications for applied sciences (phytopathology, molecular biology) mainly at the species level; besides name changes for some taxa, revised species circumscriptions and improved species identification using genetic markers have important consequences on host ranges, source inocula and risk assessment of phytopathologically important taxa. However, there are also some substantial unresolved problems which need to be addressed in the future with new data and methods. These include the systematic position of some rarely sampled taxa, the phylogenetic relationships of the main downy mildew lineages to each other, more detailed molecular studies on speciation processes to develop appropriate sound species concepts and circumscriptions, and the development of a molecular bar coding system for downy mildews enabling reliable species identification. Applying molecular methods has the potential to greatly enhance our knowledge on the overall biodiversity of downy mildews.
Over the past decade, extensive research on the wild-plant pathosystem, Lactuca serriola (prickly lettuce)–Bremia lactucae (lettuce downy mildew), has been conducted in the Czech Republic. Studies focused on pathogen occurrence and distribution,
host range, variation in symptom expression and disease severity, interactions of B. lactucae with another fungal species (Golovinomyces cichoracearum) on L. serriola, variation in resistance within natural populations of L. serriola, the structure and dynamics of virulence within populations of B. lactucae, sexual reproduction of B. lactucae, and a comparison of virulence structure and changes in B. lactucae populations occurring in wild (L. serriola) and crop (L. sativa) pathosystems. The incidence of B. lactucae on naturally growing L. serriola and other Asteraceae was recorded. Lactuca serriola was the most commonly occurring host species, followed by Sonchus oleraceus. Over the duration of these studies, the incidence of B. lactucae in L. serriola populations varied between 45–87%. Disease incidence and disease prevalence were partly related to the size, density and
different habitats of L. serriola populations. In addition to B. lactucae infection, infection by the lettuce powdery mildew fungus (Golovinomyces cichoracearum) was quite common, including co-infection. Variation in resistance to B. lactucae was studied by using ten isolates (NL and BL races with known virulence patterns) at a metapopulation level, i.e. 250 L. serriola samples representing 16 populations from the Czech Republic (CZ). Our comparisons revealed broad variation in host resistance
among host populations and also intrapopulation variability. In the CZ populations, 45 resistance phenotypes were recorded,
the most frequent were race-specific reaction patterns. Structural and temporal changes in virulence variation of B. lactucae populations on L. serriola were studied during 1998–2005. Altogether, 313 isolates of B. lactucae originating from the Czech Republic were examined for the presence of 32 virulence factors (v-factors), and 93 different
virulence phenotypes (v-phenotypes) were recorded. A study of v-factor frequency showed that common v-factors in B. lactucae populations match some of the race-specific resistance genes/factors (Dm genes or R-factors) originating from L. serriola. The highest frequency was recorded by v-factors v7, v11, v15–17, and v24–30. In contrast, v-factors (e.g. v1–4, 6, and 10)
matching Dm genes originating from L. sativa were very rare. This demonstrates the close adaptation of B. lactucae virulence to the host (L. serriola) genetic background. Temporal changes in virulence frequencies over the period were recorded. In many v-factors (v11, v14,
v16, and v25–28), fluctuations were observed, some (v14 and v17) shifting to higher frequencies, and others (v5/8 and v23)
decreasing. The occurrence of mating types was studied (1997–1999) in a set of 59 B. lactucae isolates. Both compatibility types (B1 and B2) were recorded; however the majority of the isolates (96%) were type B2. A
comparative study of B. lactucae virulence variation between the wild (L. serriola) and crop (L. sativa) pathosystems showed major differences. Migration and gene flow between both pathosystems and the potential danger of wild
B. lactucae populations for cultivated lettuce are discussed. This paper summarizes comprehensive and unique research on an oomycete
pathogen (B. lactucae) that is shared between a crop (lettuce, L. sativa) and its close wild relative (prickly lettuce, L. serriola). The data demonstrate clear evidence about race-specific interactions, variation and changes in virulence, and coevolutionary
relationships in the wild pathosystem L. serriola–B. lactucae. Conclusions contribute to the broadening and better understanding of gene-for-gene systems in natural host–pathogen populations
and their relationships to crop pathosystems.
The volatile antimicrobial substance allicin (diallylthiosulphinate) is produced in garlic when the tissues are damaged and
the substrate allicin (S-allyl-l-cysteine sulphoxide) mixes with the enzyme alliin-lyase (E.C.4.4.1.4). Allicin undergoes thiol-disulphide exchange reactions
with free thiol groups in proteins and it is thought that this is the basis of its antimicrobial action. At 50μg ml-1, allicin in garlic juice inhibited the germination of sporangia and cysts and subsequent germ tube growth by Phytophthora infestans both in vitro and in vivo on the leaf surface. Disease severity in P. infestans-infected tomato seedlings was also reduced by spraying leaves with garlic juice containing allicin over the range tested
(55–110μg ml−1) with an effectiveness ranging from approximately 45–100%. Similarly, in growth room experiments at concentrations from 50–1,000μg
ml−1, allicin in garlic juice reduced the severity of cucumber downy mildew caused by Pseudoperonospora cubensis by approximately 50–100%. These results suggest a potential for developing preparations from garlic for use in specialised
aspects of organic farming, e.g. for reducing pathogen inoculum potential and perhaps for use under glass in horticulture.
Lettuce downy mildew caused by Bremia lactucae has long been a model for understanding biotrophic oomycete–plant interactions. Initial research involved physiological and
cytological studies that have been reviewed earlier. This review provides an overview of the genetic and molecular analyses
that have occurred in the past 25years as well as perspectives on future directions. The interaction between B. lactucae and lettuce (Lactuca sativa) is determined by an extensively characterized gene-for-gene relationship. Resistance genes have been cloned from L. sativa that encode proteins similar to resistance proteins isolated from other plant species. Avirulence genes have yet to be cloned
from B. lactucae, although candidate sequences have been identified on the basis of motifs present in secreted avirulence proteins characterized
from other oomycetes. Bremia lactucae has a minimum of 7 or 8 chromosome pairs ranging in size from 3 to at least 8Mb and a set of linear polymorphic molecules
that range in size between 0.3 and 1.6Mb and are inherited in a non-Mendelian manner. Several methods indicated the genome
size of B. lactucae to be ca. 50Mb, although this is probably an underestimate, comprising approximately equal fractions of highly repeated
sequences, intermediate repeats, and low-copy sequences. The genome of B. lactucae still awaits sequencing. To date, several EST libraries have been sequenced to provide an incomplete view of the gene space.
Bremia lactucae has yet to be transformed, but regulatory sequences from it form components of transformation vectors used for other oomycetes.
Molecular technology has now advanced to the point where rapid progress is likely in determining the molecular basis of specificity,
mating type, and fungicide insensitivity.
A monoclonal antibody that recognises components of the wall of sporangia of Peronospora destructor was raised. Tests using spores of higher fungi and other species of mildew demonstrated the specificity of the monoclonal.
The antibody was used to develop lateral flow devices for sporangia of P. destructor. A competitive lateral flow format was developed which could detect onion downy mildew sporangia. Five-microliter gold anti-mouse
IgM solution pre-mixed with 10μl of P. destructor monoclonal antibody (EMA 242) proved the optimal concentration for detection of sporangia of P. destructor when applied to sample pads of lateral flow devices. Limits of approximately 500 sporangia of P. destructor could be detected by the absence of a test line on the lateral flow device within test samples. Using a scanning densitometer
improved the sensitivity of detection. Further development and validation of the test is required if it is to be used for
risk assessments of onion downy mildew in the field.
Plant pathogenic oomycetes, including biotrophic downy mildews and hemibiotrophs/necrotrophs such as Phytophthora and Pythium, cause enormous economic losses on cultivated crops. Lettuce breeders and growers face the threat of Bremia lactucae, the causal agent of lettuce downy mildew. This pathogen damages leaf tissues and lettuce heads and is also frequent on wild
Asteraceae plants. The interactions of Lactuca spp. with B. lactucae (abbr. lettuce–Bremia) display extreme variability, due to a long co-evolutionary history. For this reason, during the last 30years, the lettuce–Bremia pathosystem has been used as a model for many studies at the population, individual, organ, tissue, cellular, physiological
and molecular levels, as well as on genetic variability and the genetics of host–parasite interactions. The first part of
this review summarizes recent data on host–parasite specificity, host variability, resistance mechanisms and genetics of lettuce–Bremia interactions. The second part focuses on the development infection structures. Phenotypic expression of infection, behaviour
of B. lactucae on leaf surfaces, the process of penetration, development of primary infection structures, hyphae and haustoria are discussed
in relation to different resistance mechanisms. In the third part, the components of host resistance and the variability of
defence responses are analysed. The role of reactive oxygen species (ROS), antioxidant enzymes, nitric oxide (NO), phenolic
compounds, reorganization of cytoskeleton, electrolyte leakage, membrane damage, cell wall disruption, hypersensitive reaction
and plant energetics are discussed in relation to defence responses. In general, the extreme variability of interactions between
lettuce and Bremia, and their phenotypic expression, results from diversity of the genetic background. Different mechanisms of resistance are
conditioned by an orchestra of defence responses at the tissue, cell, and molecular levels. The various events responsible
for defence involve a complex interaction of the processes and reactions mentioned above. This review also provides an overview
on the timing of pathogen development, host pathological anatomy, cytology and physiology of lettuce–Bremia associations. The significance of these factors on the expression of different resistance mechanisms (non-host and host resistance,
race-specific and race non-specific resistance, field resistance) is discussed.
Efficient and synchronized production of infection structures of Phytophthora infestans, the causal agent of late blight of potato, was established on an artificial membrane without the host plant. Microscopic comparison of the in vitro and the in planta formed fungal structures revealed a high degree of similarity. In vitro development of infection structures enabled detailed cytological and biochemical investigations. By video microscopy the highly dynamic phenomenon of cytoplasmic migration was monitored within the living fungus. At four distinct developmental stages, hyphae, cysts, germinating cysts and appressoria, all grown in vitro, protein synthesis was analysed by comparative two-dimensional SDS-polyacrylamide gel electrophoresis. On two-dimensional gels of protein extracts of the four developmental stages a number of polypeptides were identified that showed stage-specific differences in their relative amounts. The de novo synthesis of proteins was investigated by in vivo labelling experiments. A number of polypeptides showed development-dependent expression. The majority of changes in protein synthesis occurred during germination of cysts and development of the germ tubes. In particular, at the stage of appressoria formation, the actual start of the infection process, several major polypeptides were newly synthesized.
From a set of Phytophthora infestans cDNA clones that were randomly selected from a potato- P. infestans interaction cDNA library, a relatively high proportion (5 out of 22) appeared to be derived from the same gene. The gene
was designated ric1. P. infestans contains two copies of ric1 which share 98% homology at the nucleotide-sequence level and 100% at the amino-acid level. The nucleotide sequence predicts
an open reading frame of 171 bp encoding a 57 amino-acid hydrophobic-peptide with two potential membrane-spanning domains.
The predicted peptide shows high homology to a peptide encoded by plant genes whose expression is specifically induced during
stress conditions. Southern-blot analysis of genomic DNA of several Phytophthora species indicated that most species contain ric1 homologues. During the life cycle of P. infestans, ric1 was expressed in all developmental stages but the level of expression varied. Sporangia and germinating cysts appeared to
contain only very little ric1 mRNA whereas in the mycelium and during in planta growth higher levels were detected. Subjecting the mycelium to osmotic
stress or to a high pH resulted in increased ric1 expression.
BABA, a non-protein amino acid, was used to induce resistance in grapevine against downy mildew. BABA-induced resistance was
observed in the susceptible cv. Chasselas as well as in the resistant cv. Solaris. Following BABA treatment, sporulation of
Plasmopara viticola was strongly reduced and the accumulation of stilbenes increased with time following infection. Induction of trans-piceide, trans-resveratrol and, more importantly, of trans-ɛ- and trans-δ-viniferin and trans-pterostilbene was observed in BABA-primed Chasselas. On the other hand, induction of trans-resveratrol, trans δ-viniferin and trans-pterostilbene was observed in BABA-primed Solaris. The accumulation of stilbenes in BABA-primed Solaris was much higher than
that found in BABA-primed Chasselas. Furthermore, BABA-treatment of Solaris led to a rapid increase in transcript levels of
three genes involved in the phenylpropanoid pathway: phenylalanine ammonia lyase, cinnamate-4-hydroxylase and stilbene synthase.
BABA-primed Chasselas showed increased transcript levels for cinnamate-4-hydroxylase and stilbene synthase. Here we show that
pre-treatment of a susceptible grapevine cultivar with BABA prior to infection with P. viticola primed the accumulation of specific phytoalexins that are undetectable in non-BABA-primed plants. As a result, the susceptible
cultivar became more resistant to downy mildew.
Several plant pathogenic oomycetes have been under investigation using modern molecular approaches. Genome sequencing and
annotations are underway or near to completion for some of the species. Pathogen-associated molecular pattern molecules (PAMPs)
and effector molecules perform inter- and intracellular tasks as adaptation factors and manipulators of the defence network.
Hundreds of secreted putative effectors have been discovered and conserved molecular patterns such as RXLR and EER motifs
have been identified and used for classifications. PAMPs and effectors are recognized directly or indirectly by the pattern
recognition receptors at the cell surface including receptor-like kinases and receptor-like proteins, and/or by nucleotide
binding site–leucine rich repeat proteins within the cytoplasm. The current knowledge of effectors, immune receptors and the
defence network, will help us understand the ‘intricate genetic dance’ between the oomycete pathogens and their hosts. This
review concentrates on the recent findings in oomycete-plant interactions.
Four carboxylic acid amide (CAA) fungicides, mandipropamid (MPD), dimethomorph (DMM), benthiavalicarb (BENT) and iprovalicarb
(IPRO) were examined for their effects on various developmental stages of Bremia lactucae, the causal agent of downy mildew in lettuce, in vitro and in planta. Spore germination in vitro or on leaf surfaces was inhibited
by all CAA fungicides (technical or formulated). MPD was more effective in suppressing germination than DMM or BENT, whereas
IPRO was least effective. CAA induced no disruption of F-actin microfilament organisation in germinating spores of B. lactucae. CAA applied to germinating spores in vitro prevented further extension of the germ tubes. When applied to germinated spores
on the leaf surface they prevented penetration. Preventive application of CAA to intact plants inhibited infection. MPD was
more effective in suppressing infection than DMM or BENT, whereas IPRO was least effective. Curative application was effective
at ≤3h post-inoculation (hpi) but not at ≥18hpi. CAA (except IPRO) applied to upper leaf surfaces inhibited spore germination
on the lower surface and hence reduced infection. CAA suppressed sporulation of B. lactucae on floating leaf discs and when sprayed onto infected plants two days before onset of sporulation. BENT and DMM were more
effective in suppressing sporulation than MPD or IPRO. Epidemics of downy mildew in shade-house grown lettuce were suppressed
by CAA. A single spray applied to five-leaf plants before transplanting controlled the disease for 50days. The results suggest
that CAA are effective inhibitors of spore germination and therefore should be used as preventive agents against downy mildew
of lettuce caused by B. lactucae.
This review provides a summary of recent examples of interspecific hybridisation within the oomycetous genus Phytophthora. Species hybrids either created in the laboratory or evolved in natural environments are discussed in association with evolutionary
issues and possible threats they may pose to agriculture, horticulture and forestry. It is suggested that sustainable control
of such hybrids will depend on the better understanding of temporal and spatial aspects of genetic mechanisms and environmental
factors that lead to the hybridisation process and thus the genetic diversity in Phytophthora populations.
Plants are constantly beset by pathogenic organisms. To successfully infect their hosts, plant pathogens secrete effector proteins, many of which are translocated to the inside of the host cell where they manipulate normal physiological processes and undermine host defense. The way by which effectors cross the frontier to reach the inside of the host cell varies among different classes of pathogens. For oomycete plant pathogens – like the potato late blight pathogen Phytophthora infestans – it has been shown that effector translocation to the host cell cytoplasm is dependent on conserved amino acid motifs that are present in the N-terminal part of effector proteins. One of these motifs, known as the RXLR motif, has a strong resemblance with a host translocation motif found in effectors secreted by Plasmodium species. These malaria parasites, that reside inside specialized vacuoles in red blood cells, make use of a specific protein translocation complex to export effectors from the vacuole into the red blood cell. Whether or not also oomycete RXLR effectors require a translocation complex to cross the frontier is still under investigation. For one P. infestans RXLR effector named IPI-O we have found a potential host target that could play a role in establishing the first contact between this effector and the host cell. This membrane spanning lectin receptor kinase, LecRK-I.9, interacts with IPI-O via the tripeptide RGD that overlaps with the RXLR motif. In animals, RGD is a well-known cell adhesion motif; it binds to integrins, which are membrane receptors that regulate many cellular processes and which can be hijacked by pathogens for either effector translocation or pathogen entry into host cells.
Potato (Solanum tuberosum) is the world's third-largest food crop. It severely suffers from late blight, a devastating disease caused by Phytophthora infestans. This oomycete pathogen secretes host-translocated RXLR effectors that include avirulence (AVR) proteins, which are targeted by resistance (R) proteins from wild Solanum species. Most Solanum R genes appear to have coevolved with P. infestans at its center of origin in central Mexico. Various R and Avr genes were recently cloned, and here we catalog characterized R-AVR pairs. We describe the mechanisms that P. infestans employs for evading R protein recognition and discuss partial resistance and partial virulence phenotypes in the context of our knowledge of effector diversity and activity. Genome-wide catalogs of P. infestans effectors are available, enabling effectoromics approaches that accelerate R gene cloning and specificity profiling. Engineering R genes with expanded pathogen recognition has also become possible. Importantly, monitoring effector allelic diversity in pathogen populations can assist in R gene deployment in agriculture.
The fungal pathogen Phytophthora infestans is the causal organism of late blight, one of the most devastating diseases of potato. In the past, various aspects of the potato-P. infestans interaction have been studied extensively. In this paper we briefly review the current knowledge of the molecular events associated with the interaction and in addition we discuss a new approach for analyzing the molecular basis of pathogenicity of P. infestans. © 1992 Koninklijke Nederlandse Planteziektenkundige Vereniging.
Induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is essential for the biosynthesis of sesquiterpenoid phytoalexins and steroid derivatives in Solanaceous plants following stresses imposed by wounding and pathogen infection. To better understand this complex step in stress-responsive isoprenoid synthesis, we isolated three classes of cDNAS encoding HMGR (hmg1, hmg2, and hmg3) from a potato tuber library using a probe derived from an Arabidopsis HMGR cDNA. The potato cDNAs had extensive homology in portions of the protein coding regions but had low homology in the 3' untranslated regions. RNA gel blot analyses using gene-specific probes showed that hmg1 was strongly induced in tuber tissue by wounding, but the wound induction was strongly suppressed by treatment of the tissue with the fungal elicitor arachidonic acid or by inoculation with an incompatible or compatible race of the fungal pathogen Phytophtora infestans. The hmg2 and hmg3 mRNAs also accumulated in response to wounding, but in contrast to hmg1, these mRNAs were strongly enhanced by arachidonic acid or inoculation. Inoculation with a compatible race of P. infestans resulted in similar patterns in HMGR gene expression of hmg2 and hmg3 except that the magnitude and rate of the changes in mRNA levels were reduced relative to the incompatible interaction. The differential regulation of members of the HMGR gene family may explain in part the previously reported changes in HMGR enzyme activities following wounding and elicitor treatment. The suppression of hmg1 and the enhancement of hmg2 and hmg3 transcript levels following elicitor treatment or inoculation with the incompatible race parallel the suppression in steroid and stimulation of sesquiterpenoid accumulations observed in earlier investigations. The results are discussed in relation to the hypothesis that there are discrete organizational channels for sterol and sesquiterpene biosynthesis in potato and other Solanaceous species.
An in planta induced gene of Phytophthora infestans (the causal organism of potato late blight) was selected from a genomic library by differential hybridization using labelled cDNA derived from poly(A)+ RNA of P. infestans grown in vitro and labelled cDNA made from potato-P. infestans interaction poly(A)+ RNA as probes. Sequence analysis showed that the gene codes for ubiquitin, a highly conserved protein which plays an important role in several cellular processes. The structure of the polyubiquitin gene (designated ubi3R) is consistent with the structure of other known polyubiquitin genes. It consists of three repeats in a head-to-tail arrangement without intervening sequences, each encoding a ubiquitin unit of 76 amino acids. The last ubiquitin unit is followed by an extra asparagine residue at the carboxy-terminal end. Northern and Southern blot analyses revealed that the polyubiquitin gene is a member of a multigene family, all genes of which show induced expression in planta.
Polar axis formation and polar axis stabilization (or fixation) can be separated and analyzed in synchronously developing zygotes of the brown alga Fucus. Extensive experimental evidence points to a role for both the cytoskeleton and the extracellular matrix (ECM) in the process of axis fixation in Fucus. A structural complex composed of the cytoskeleton and the ECM has been postulated to stabilize membrane asymmetries generated as a result of axis-forming vectors. This axis stabilizing complex (ASC) may take the form of transmembrane connections between the cytoskeleton on the cytoplasmic face and the ECM on the external side of the plasma membrane, similar to focal contacts in animal cells. At present we know of two components in the proposed ASC of Fucus: an adhesive sulfated glycoprotein which is localized in the ECM, and an actin network which is localized on the adjoining cytoplasmic face. This preliminary report describes evidence for the presence of molecules in two-celled Fucus embryos that are similar to those found in focal contacts in animal cells, i.e. vinculin, integrin and vitronectin. However, their localization and interaction with each other relative to the polar axis has yet to be determined. These initial observations will provide the basis to pursue further an analysis of these components in the process of polar axis fixation.
Soybean (Glycine max [L.] Merr. cv. Mandarin) root cells (SB-1 cell line) grown in suspension culture containing Glycyl-Arginyl-Glycyl-Aspartyl-Seryl-Proline (GRGDSP) (0.25 mg/ml), a synthetic peptide containing the RGD sequence found in many extracellular matrix adhesive proteins, demonstrated (a) significantly enhanced growth rate, and (b) aberrant cell wall/plasma membrane interactions and organization. Substitution of the Asp (D) by a Glu (E) amino acid in the hexapeptide, or inversion of the RGD sequence to GDR, abolished the morphological and growth effects observed for GRGDSP in plant cells. Immunoblots, which were prepared from beta-octylglucoside extracts of whole soybean cells and protoplasts, probed with polyclonal antibodies raised against human vitronectin receptor (hVNR) complex, demonstrated a single band with an apparent molecular mass of 70-72 kD. Chromatography of beta-octylglucoside extracts of SB-1 cells on a Gly-Arg-Gly-Asp-Ser-Pro-Lys-Sepharose affinity column demonstrated the retention of a single 70-72 kD polypeptide that reacted specifically with anti-hVNR antiserum. In contradistinction, no cross-reactivity was observed with antifibronectin receptor antiserum. Epifluorescence microscopy of whole soybean cells, after moderate treatment with pectinase, demonstrated punctate fluorescent patches at the cell membrane/wall boundary when probed with anti-hVNR and rhodamine-derivatized secondary antibodies. We propose that coordination and control of plant cell division and proper cell wall biosynthesis may be mediated by an RGD-dependent recognition system in which RGD binding protein(s) promote cell membrane-cell wall attachment.
I report on the isolation, structural analysis, and in vivo expression patterns of a fungal calmodulin gene. The gene is intronless and encodes a protein of 148 amino acid residues that is 92% homologous with vertebrate calmodulins. Through S1 nuclease transcript mapping, it was determined that the cloned gene (a) is transcribed in vivo, (b) has a 5'-untranslated region of about 400 nucleotides, and (c) has a 3'-untranslated end of about 300 nucleotides. Southern blot hybridization analysis of the genomic DNA and the cloned gene provide evidence for the existence of only one type of calmodulin gene in the organism. The amino acid sequence deduced from the DNA sequence shows that Achlya klebsiana calmodulin has amino acid substitutions that are a mix of those seen in calmodulins from invertebrates such as Drosophila and trypanosome when compared to mammalian calmodulins. Not surprisingly, it has less resemblance to calmodulins from Saccharomyces and Dictyostelium.
A single genomic clone (14 kb) isolated from bean (Phaseolus vulgaris L.) contains two genes that encode glycine-rich proteins. These genes are present as single copies in the genome, are separated by 2.85 kb and encode transcripts of 1.8 kb and 1.0 kb respectively. The encoded proteins contain 60% glycine and have amino-terminal signal peptides. The 1.8 kb transcript is present in young hypocotyls and in ovary tissue. Excision-wounding transiently induced this transcript in old, but not in young hypocotyl tissue. Antibodies raised against regions of the glycine-rich protein 1.8, expressed as a lacZ fusion protein in bacteria, react with a protein of 53 kd in a protein fraction extracted from cell walls of bean ovaries. Tissue imprints of bean ovaries treated with anti-glycine-rich protein antibodies showed that the glycine-rich protein was distributed in a regular pattern of small, highly localized discrete sites. The immunoreactive regions correspond to the pattern of vascular tissue in the pod. In young hypocotyls, glycine-rich protein is present at four pairs of discrete sites symmetrically arranged on the inner side of the vascular ring. These results suggest a close relationship between glycine-rich proteins and development of the vascular system.
The major surface glycoprotein of Leishmania promastigotes, gp63, was isolated and reconstituted into a lipid membrane immobilized on the surface of 5-micron-diameter silica beads. These beads bound to the macrophage (MO), and the extent of binding correlated with the density of gp63 on the bead. The bead thus facilitated analysis of the binding specificity of a single ligand, gp63, without contribution from other molecules present on the surface of intact promastigotes. Plating of MO onto substrates coated with antibodies directed against several cell surface receptors indicated that the complement receptor CR3 was necessary for binding gp63. CR3 recognizes a portion of C3 that contains the sequence R G D. Since gp63 also contains such a sequence, we tested the ability of a synthetic peptide based on the R G D-containing region of gp63 to inhibit the binding of gp63 beads. The R G D-containing peptide from gp63 inhibited the binding of both gp63 beads and EC3bi to MO. Similarly, peptides previously shown to inhibit the binding of C3bi also inhibited the attachment of gp63 beads. The synthetic peptide from the R G D region of gp63 also reduced the binding of intact promastigotes to MO. These results indicate that gp63 binds directly to CR3.
A new method for identifying secretory signal sequences and for predicting the site of cleavage between a signal sequence
and the mature exported protein is described. The predictive accuracy is estimated to be around 75–80% for both prokaryotic
and eukaryotic proteins.
The extracellular matrix (ECM) has been implicated in the primary developmental processes of many organisms. A family of secretory adhesive glycoproteins called substrate adhesion molecules (SAMs) is believed to confer these dynamic capabilities to the ECM in animals. In this paper, we report the existence of SAM-like genes and gene products in flowering plants. Hybridizations with a human vitronectin cDNA probe and genomic DNA from broad bean, soybean, and tomato revealed vitronectin-like sequences. Human vitronectin antibodies cross-react with a 55-kilodalton protein in leaf and root protein extracts from lily, broad bean, soybean, and tomato. In addition, immunocytochemical staining of frozen sections of lily leaf and broad bean gynoecium demonstrated that vitronectin-like proteins were localized to the ECM on the cell surface, with the most intense labeling residing in the transmitting tract of broad bean gynoecium.
In French bean (Phaseolus vulgarisL.), the glycine-rich wall protein GRP 1.8 is specifically synthesized in protoxylem tracheary elements of the vascular system. A 494 bp upstream promoter fragment of the gene encoding GRP 1.8 was isolated and translationally fused to the beta-glucuronidase reporter gene. Transgenic tobacco plants containing this construct expressed the gene in vascular tissue of roots, stems, leaves and flowers. The gene was developmentally expressed during differentiation of both primary and secondary vascular tissue and was also rapidly induced (in < 30 min) after excision-wounding of young stems. This wound response is more rapid than in bean hypocotyls, indicating possible differences between the activation mechanism for glycine-rich protein gene expression in wounded bean and tobacco. Only a subset of cells were found to participate in the wound response. In young stems, the GRP wound induction was localized in pith parenchyma cells adjacent to the wound surface, where vessel regeneration is known to occur. Thus, a promoter fragment of 494 bp, including 427 bp upstream from the transcription start site, contains information for tissue-specific and wound-induced gene regulation. The cell-type specificity of expression suggests that the GRP 1.8 promoter is regulated by very specific developmental and environmental signals.
An antibody against glycine-rich protein 1.8 of bean (Phaseolus vulgaris L.) was used for immunogold/silver localization of the protein in different organs of the plant. In hypocotyls, ovaries, and seed coats, the protein was found specifically in xylem cells of the vascular tissue. In hypocotyls, only protoxylem cells were labeled with the antibody, which indicates a remarkable cell-type specificity for accumulation of this cell wall protein. In mature hypocotyls, the protein was restricted to the same subset of xylem cells but was no longer detected on tissue prints, where a positive antibody reaction depends on the transfer of soluble material from plant tissue to the nitrocellulose filter. This indicates that the glycine-rich protein is insolubilized in the cell wall during development. In longitudinal sections of tracheary elements of young hypocotyls and seed coats, the antibody stained a pattern very similar to that of the lignified secondary thickenings of the cell wall, which suggests a close functional relationship between glycine-rich protein and lignin deposition during cell wall biogenesis in protoxylem cells.
Infection of potato leaves with the fungal pathogen Phytophthora infestans (Pi) resulted in the rapid stimulation of phenylpropanoid metabolism. Increases in the activities of several mRNAs, including those encoding phenylalanine ammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL), were detectable within a few hours postinoculation, as demonstrated by two-dimensional gel electrophoresis of proteins synthesized in vitro. This effect was closely mimicked by application of Pi culture filtrate through cut leaf stems. PAL and 4CL mRNA activities were also rapidly and transiently induced in potato cell suspension cultures by treatments with Pi culture filtrate or arachidonic acid. This induction was exploited to generate cDNA probes complementary to PAL and 4CL mRNAs. Blot hybridizations using these probes revealed almost immediate, transient and coordinate increases in the transcription rates and subsequent changes in the amounts of PAL and 4CL mRNAs in leaves treated with Pi culture filtrate. Similar changes in the mRNA amounts were found in infected leaves of potato cultivars carrying resistance genes R1 (cv Datura) or R4 (cv Isola), independent of whether a virulent or an avirulent Pi pathotype was used for inoculation. These results are discussed in relation to recent cytological observations with the same potato cultivars and Pi pathotypes.
The response of potato (Solanum tuberosum) leaf tissue to infection by the late-blight fungus Phytophthora infestans (Pi) is very rapid. Inoculation of either the lower or the upper side of a potato leaf with Pi zoospores under proper environmental conditions is followed by germination of the fungus within minutes and penetration of hyphae through stomata or subsidiary cells, or occasionally between epidermal cells (Cuypers and Hahlbrock 1988). The earliest readily detectable responses of immediately affected plant cells are callose deposition and the accumulation of phenolic compounds in the cell wall. Both effects are clearly visible about 2–3 h postinoculation. The accumulation of cell wa11-associated phenolics at this early stage is an indication of hypersensitive cell death and is detected most easily by autofluorescence of the cells under UV light (Fig. 1).
Soybean (Glycine max [L.] Merr. cv. Mandarin) root cells (SB-1 cell line) grown in suspension culture containing Glycyl-Arginyl-Glycyl-Aspartyl-Seryl-Proline (GRGDSP) (0.25 mg/ml), a synthetic peptide containing the RGD sequence found in many extracellular matrix adhesive proteins, demonstrated (a) significantly enhanced growth rate, and (b) aberrant cell wall/plasma membrane interactions and organization. Substitution of the Asp (D) by a Glu (E) amino acid in the hexapeptide, or inversion of the RGD sequence to GDR, abolished the morphological and growth effects observed for GRGDSP in plant cells. Immunoblots, which were prepared from beta-octylglucoside extracts of whole soybean cells and protoplasts, probed with polyclonal antibodies raised against human vitronectin receptor (hVNR) complex, demonstrated a single band with an apparent molecular mass of 70-72 kD. Chromatography of beta-octylglucoside extracts of SB-1 cells on a Gly-Arg-Gly-Asp-Ser-Pro-Lys-Sepharose affinity column demonstrated the retention of a single 70-72 kD polypeptide that reacted specifically with anti-hVNR antiserum. In contradistinction, no cross-reactivity was observed with antifibronectin receptor antiserum. Epifluorescence microscopy of whole soybean cells, after moderate treatment with pectinase, demonstrated punctate fluorescent patches at the cell membrane/wall boundary when probed with anti-hVNR and rhodamine-derivatized secondary antibodies. We propose that coordination and control of plant cell division and proper cell wall biosynthesis may be mediated by an RGD-dependent recognition system in which RGD binding protein(s) promote cell membrane-cell wall attachment.
In order to isolate in planta-induced genes encoding putative pathogenicity factors of the late blight fungus Phytophthora infestans, a genomic library was differentially screened. For the differential hybridization, labeled first-strand cDNA synthesized on mRNA isolated from P. infestans-infected potato leaves and on mRNA isolated from the fungus grown in vitro were used as probes. This screening resulted in the isolation of the P. infestans calmodulin gene. The gene, designated calA, contains an open reading frame of 447 base pairs without introns and is unique in the P. infestans genome. The predicted amino acid sequence is 89.9-94.6% identical to calmodulins from higher eukaryotes, whereas the identity to calmodulins of higher fungi is significantly less (60.8-85.1%). Expression studies revealed that the P. infestans calA gene is constitutively expressed in in vitro grown mycelium. However, during pathogenesis on potato the level of P. infestans calmodulin mRNA is increased approximately fivefold.
We have examined the expression of the petunia (Petunia hybrida) glycine-rich protein-1 (ptGRP1) gene product using an antibody raised against a synthetic peptide comprising amino acids 22 through 36 of the mature ptGRP1 protein. This antibody recognizes a single protein of 23 kilodaltons. Cell fractionation studies showed that, as predicted (CM Condit, RB Meagher [1986] Nature 323: 178-181), ptGRP1 is most likely localized in the cell wall. In addition, it was found that (extractable) ptGRP1 is present in much higher abundance in unexpanded than in fully expanded tissue, with highest levels of accumulation in the bud. This same developmentally regulated pattern of protein expression was found in all varieties of petunia tested. In addition, tissue blots of petunia stem sections showed that ptGRP1 is localized to within the vascular tissue (to at least the phloem or cambium) and to either the epidermal cells or to a layer of collenchyma cells directly below the epidermis. Localization of ptGRP1 antigen in these cell types is shown to occur at different times in the overall development of the plant and at different quantitative levels.
Ubiquitin has been suggested to play a key role in a wide variety of essential cellular functions ranging from differential regulation of gene expression to protein degradation. Recent studies on natural and synthetic ubiquitin gene fusions have led to important discoveries concerning novel functions for the ubiquitin system in cells, mechanisms of proteolytic processing, and the development of a ubiquitin fusion technology for augmenting the expression of heterologous gene products in bacteria and yeast. Furthermore, studies involving site-directed mutagenesis and two-dimensional NMR have proven ubiquitin to be an excellent model for protein engineering and have led to important discoveries concerning its mechanism of action in ATP-dependent proteolysis. Finally, the recent identification and characterization of ubiquitin carboxyl extension proteins as ribosomal proteins has opened up an even newer area of ubiquitin-related research and has helped to explain the mechanisms involved in increasing the expression of heterologous gene products made as ubiquitin fusions.
Cells of tobacco adapted to grow in high concentrations of NaCl develop tight zones of adhesion between the plasma membrane and cell wall, revealed by concave plasmolysis in osmotic solutions. Unadapted cells exhibit mostly convex plasmolysis and exhibit little or no adhesive character. Wall-less protoplasts isolated from the adapted cells retain the complementary adhesive character and adhere tightly to each other, whereas protoplasts from unadapted cells do not. The hexapeptide gly-arg-gly-asp-ser-pro, in which the arg-gly-asp represents the integrin-binding domain of several animal extracellular matrix proteins, specifically blocks adhesion of the protoplasts. A control hexapeptide, gly-arg-gly-glu-ser-pro, is ineffective in blocking adhesion. Tobacco proteins immunologically related to human vitronectin were found in cell walls and membranes of unadapted and NaCl-adapted cells, but the total extractable vitronectin-like protein was enriched in the adapted cells. Tobacco proteins immunologically related to human fibronectin were found in membranes and cell walls of NaCl-adapted cells but not in those from unadapted cells. Our observations indicate that plant cells possess cell-matrix adhesion complexes similar to animal cells, and these adhesion complexes accumulate in growth-limited cells adapted to saline stress.
A computer program that progressively evaluates the hydrophilicity and hydrophobicity of a protein along its amino acid sequence has been devised. For this purpose, a hydropathy scale has been composed wherein the hydrophilic and hydrophobic properties of each of the 20 amino acid side-chains is taken into consideration. The scale is based on an amalgam of experimental observations derived from the literature. The program uses a moving-segment approach that continuously determines the average hydropathy within a segment of predetermined length as it advances through the sequence. The consecutive scores are plotted from the amino to the carboxy terminus. At the same time, a midpoint line is printed that corresponds to the grand average of the hydropathy of the amino acid compositions found in most of the sequenced proteins. In the case of soluble, globular proteins there is a remarkable correspondence between the interior portions of their sequence and the regions appearing on the hydrophobic side of the midpoint line, as well as the exterior portions and the regions on the hydrophilic side. The correlation was demonstrated by comparisons between the plotted values and known structures determined by crystallography. In the case of membrane-bound proteins, the portions of their sequences that are located within the lipid bilayer are also clearly delineated by large uninterrupted areas on the hydrophobic side of the midpoint line. As such, the membrane-spanning segments of these proteins can be identified by this procedure. Although the method is not unique and embodies principles that have long been appreciated, its simplicity and its graphic nature make it a very useful tool for the evaluation of protein structures.
Fruiting in the basidiomycete Schizophyllum commune is accompanied by transcription of a small number of specific genes that produce abundant mRNAs. These fruiting genes are regulated by the mating-type genes MATA and MATB, as well as by other genes such as FBF and THN, but also by the developmental stage of the mycelium. The developmentally regulated genes characterized thus far belong to gene families and code for excreted proteins. One gene family encodes hydrophobins, small hydrophobic cysteine-rich proteins which are excreted into the culture medium by submerged hyphae but accumulate in the walls of emergent hyphae where they form highly-insoluble complexes. The genes Sc1 and Sc4 of this family are only active in the dikaryon and the encoded hydrophobins typically accumulate in hyphal walls of developing fruit bodies. Another member of this family, Sc3, is also expressed in monokaryons and the encoded hydrophobin accumulates in walls of individually growing aerial hyphae. The effects of mutations in the FBF and THN genes indicate that these differentially regulated hydrophobins may play a key role in the emergence of aerial structures while different hydrophobins may determine specific hyphal-surface properties.
Plant pathogens produce pathogenicity factors which enable them to parasitize and colonize their host. A strategy for identifying pathogenicity factors involves the isolation and characterization of genes encoding these factors. Potential pathogenicity genes are genes whose expression is induced during pathogenesis. In order to isolate such genes of the late blight Fungus Phytophthora infestans , a genomic library of P. infestans DNA was differentially hybridized using labelled first strand cDNA probes synthesized on mRNA isolated from P. infestans infected potato leaves and on mRNA isolated from the fungus grown on a basic medium in culture. In total, nine distinct in planta induced (ipi) genes were selected. Expression studies revealed that the mRNA levels of seven of these genes, ipiA, ipi C, ipiD, ipiJ1, ipiJ2, ipiN and ipi Q increased 5-10 fold during colonization. The two other genes, ipi B and ipiO, showed a transient expression pattern with the highest mRNA levels in the early stages of infection.
A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.
The TRP1 gene was isolated from the genome of Phytophthora parasitica. It encodes bifunctional enzyme of the tryptophan biosynthetic pathway indole-3-glycerolphosphate synthase-N-(5'-phosphoribosyl)anthranilate isomerase (IGPS-PRAI). The gene was localized and sequenced using random in vitro insertions of omega interposon. The domain structure of the protein product was found to be similar to that of enteric bacteria but different from the structure of homologous enzymes in fungi. Two introns in the IGPS domain were found. This is unique in eukaryotic IGPS-encoding genes so far sequenced. Comparative analysis of the primary structure of IGPS and PRAI domains [neighbor-joining method of Saitou and Nei, Mol. Biol. Evol. 44 (1987) 406-425] confirmed a large phylogenetic distance of TRP1 from corresponding fungal genes. In the resulting distance tree Phytophthora sequences are located outside of the cluster which encompasses all known homologous proteins from fungi indicating that the lineage of oomycetes took a separate course of development before speciation within the fungal line of descent began. Two of the oligopeptide insertions engineered into the F domain of the protein product did not abolish the enzymatic activity of the protein.
Actin (ACT) in Phytophthora infestans is encoded by at least two genes, in contrast to unicellular and other filamentous fungi where there is a single gene. These genes (designated actA and actB) have been isolated from a genomic library of P. infestans. The complete nucleotide sequence of both genes has been determined. Unlike the actin-encoding genes (act) of other filamentous fungi, no introns are obvious in the coding region, a feature shared with the act genes of certain protists. Northern blotting and primer extension studies of the mRNA show that actA and actB are actively transcribed in mycelium, sporangia and germinating cysts but only at a low level in the case of actB. Both genes display bias in their codon usage. This is more extreme in actA. The deduced ACTB protein is strikingly similar to that of the Phytophthora megasperma actin and is more diverged from other actins than ACTA.
This paper reports the cloning and sequencing of a Phytophthora megasperma actin gene. Sequence analysis revealed that the gene does not contain introns and encodes a putative actin protein remarkably diverged from any other known actins. Genomic Southern analysis indicates that there is a single actin gene per haploid genome, which is actively transcribed in vivo, as revealed by S1 mapping of the transcripts. The transcripts are heterogeneous in size because they have different 3' termini, none of which contains the conventional AATAAA polyadenylation signal.
Germinating spores of the obligate pathogen Bremia lactucae (lettuce downy mildew) contain several unusually abundant species of mRNA. Thirty-nine cDNA clones corresponding to prevalent transcripts were isolated from a library synthesized using poly(A)+ RNA from germinating spores; these clones represented only five distinct classes. Each corresponding mRNA accounted for from 0.4 to 9 percent by mass of poly(A)+ RNA from germinating spores and together represented greater than 20 percent of the mRNA. The expression of the corresponding genes, and a gene encoding Hsp70, was analyzed in spores during germination and during growth in planta. The Hsp70 mRNA and mRNA from one abundant cDNA clone (ham34) were expressed constitutively. Two clones (ham9 and ham12) hybridized only to mRNA from spores and germinating spores. Two clones (ham37 and ham27) showed hybridization specific to germinating spores. Quantification of the number of genes homologous to each cDNA clone indicated that four clones corresponded to one or two copies per haploid genome, and one hybridized to an approximately 11-member family of genes. A sequence of the gene corresponding to ham34 was obtained to investigate its function and to identify sequences conferring high levels of gene expression for use in constructing vectors for the transformation of B. lactucae.
The structure, genomic organization, and temporal pattern of activation of a gene encoding a pathogenesis-related protein (PR1) in potato (Solanum tuberosum) have been analyzed. The gene is rapidly activated in leaves from the potato cultivar Datura, containing the resistance gene R1, in both compatible and incompatible interactions with appropriate races of the late-blight fungus Phytophthora infestans. Activation is also observed in leaves treated with fungal elicitor. The gene occurs in multiple, very similar copies and encodes a polypeptide (Mr = 25,054; pI = 5.5) that is classified as a PR protein by several criteria. Small fragments with great sequence similarity to portions of the two exons were found closely linked to the expressed gene, which altogether represents a simple case of genome organization in potato. The coding sequence of the prp1 gene and the deduced amino acid sequence are strikingly similar to the corresponding sequences of a 26-kDa heat shock protein from soybean.
During the course of whooping cough, Bordetella pertussis interacts with alveolar macrophages and other leukocytes on the respiratory epithelium. We report here mechanisms by which these bacteria adhere to human macrophages in vitro. Whole bacteria adhere by means of two proteins, filamentous hemagglutinin (FHA) and pertussis toxin, either of which is sufficient to mediate adherence. FHA interacts with two classes of molecules on macrophages, galactose-containing glycoconjugates and the integrin CR3 (alpha M beta 2, CD11b/CD18). The interaction between CR3 and FHA involves recognition of the Arg-Gly-Asp (RGD) sequence at positions 1097-1099 in FHA. This study demonstrates that bacterial adherence can be based on the interaction of a bacterial adhesin RGD sequence with an integrin and that bacterial adhesins can have multiple binding sites characteristic of eukaryotic extracellular matrix proteins.
Discoidin I, a developmentally regulated lectin in Dictyostelium discoideum, has been implicated in cell-substratum adhesion and ordered cell migration during aggregation. This depends on the cell binding site of discoidin I, which is distinct from its carbohydrate binding site. We have isolated a receptor for the cell binding site by affinity chromatography. The receptor binds immobilized discoidin I in the presence of 0.3 M galactose and can be eluted with gly-arg-gly-asp-his-asp, a synthetic peptide the sequence of which is found in discoidin I, and which blocks cell migration into aggregates. The receptor is a developmentally regulated cell-surface glycoprotein of apparent Mr approximately 67,000. Univalent antibodies specific for this glycoprotein block aggregation.
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In this paper, we introduce a computer algorithm which can be used to predict the topological features of a protein directly from its primary amino acid sequence. The computer program generates values for surface accessibility parameters and combines these values with those obtained for regional backbone flexibility and predicted secondary structure. The output of this algorithm, the antigenic index, is used to create a linear surface contour profile of the protein. Because most, if not all, antigenic sites are located within surface exposed regions of a protein, the program offers a reliable means of predicting potential antigenic determinants. We have tested the ability of this program to generate accurate surface contour profiles and predict antigenic sites from the linear amino acid sequences of well-characterized proteins and found a strong correlation between the predictions of the antigenic index and known structural and biological data.
The amino acid sequence RGD (arginine-glycine-aspartic acid) is highly conserved in the VP1 protein of foot-and-mouth disease virus (FMDV), despite being situated in the immunodominant hypervariable region between amino acids 135 and 160. RGD-containing proteins are known to be important in promoting cell attachment in several different systems, and we report here that synthetic peptides containing this sequence are able to inhibit attachment of the virus to baby hamster kidney (BHK) cells. Inhibition was dose-dependent and could be reversed on removal of the peptide. A synthetic peptide corresponding to a portion of the same hypervariable region but not containing the RGD sequence did not inhibit virus attachment under the same conditions. Antibody against the RGD region of VP1 blocked attachment of the virus to BHK cells, and neutralizing monoclonal antibodies, which neutralize virus by preventing cell attachment, were blocked by RGD-containing peptides from binding virus in an ELISA test. Cleavage of the C-terminal region of virus VP1 in situ with proteolytic enzymes reduced cell attachment, and antiserum against a peptide corresponding to this region was also able to inhibit attachment of virus to BHK cells. These results indicate that the amino acid sequence RGD at positions 145 to 147 and amino acids from the C-terminal region of VP1 (positions 203 to 213) contribute to the cell attachment site on FMDV for BHK cells.
A gene encoding a protein homologous to a 70-kDa heat-shock protein (Hsp70) was isolated from Bremia lactucae and its structure and pattern of expression were determined. This is the first report on the structure of a protein-coding gene from an Oomycete fungus. The cloned gene is a member of a small multigene family. The level of hsp70 mRNA in germlings increases from a low constitutive level in response to heat or cold treatment. A high level of the mRNA is also detected in spores. The hsp70 gene is expressed as a primary transcript of 2241 nucleotides (nt) and contains a continuous open reading frame of 2025 nt. Near the C terminus of the coding sequence is an unusual region that contains repeated enhancer-like sequences. This insert has not been described in other hsp70 genes and is not an intron. Upstream from the 5' terminus of the mRNA are multiple CCAAT motifs, a sequence similar to a consensus heat-shock regulatory element, and an A + T-rich putative 'TATA' box. A canonical polyadenylation recognition sequence is present downstream from the coding sequence. The deduced amino acid sequence is equally similar to yeast and maize Hsp70, providing further evidence of the dissimilarity between Oomycetes and true fungi. The cloning of this gene is part of our strategy to develop a transformation system for B. lactucae.
Many adhesive interactions are mediated by Arg-Gly-Asp (RGD) sequences within adhesive proteins. Such RGD sequences are frequently
recognized by structurally related heterodimers that are members of the integrin family of adhesion receptors. A region was
found in the platelet RGD receptor, gpIIb/IIIa, to which an RGD peptide becomes chemically cross-linked. This region corresponds
to residues 109 to 171 of gpIIIa. This segment is conserved among the beta subunits of the integrins (76 percent identity
of sequence), indicating that it may play a role in the adhesive functions of this family of receptors.
A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described. DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers. Over 70% of the precursor triphosphate is routinely incorporated into complementary DNA, and specific activities of over 10(9) dpm/microgram of DNA can be obtained using relatively small amounts of precursor. These "oligolabeled" DNA fragments serve as efficient probes in filter hybridization experiments.
The University of Wisconsin Genetics Computer Group (UWGCG) has been organized to develop computational tools for the analysis
and publication of biological sequence data. A group of programs that will interact with each research-article has been developed
for the Digital Equipment Corporation VAX computer using the VMS operating system. The programs available and the conditions
for transfer are described.
5′-Noncoding sequences have been tabulated for 211 messenger R'JAs from higher eukaryotic cells. The 5′-proximal AUG triplet
serves as the initiator codon in 95% of the mRNAs examined. The most conspicuous conserved feature is the presence of a purine
(most often A) three nucleotides upstream from the AUG initiator codon; only 6 of the mRNAs in the survey have a pyrimidine
in that position. There is a predominance of C in positions −1, −2, −4 and −5, just upstream from the initiator codon. The
sequence (G) thus emerges as a consensus sequence for eukaryotic initiation sites. The extent to which the ribosome binding site in
a given mRNA matches the −1 to −5 consensus sequence varies: more than half of the mRNAs in the tabulation have 3 or 4 nucleotides
in common with the CCACC consensus, but only ten mRNAs conform perfectly.
Transcription of at least one member of the potato prp1 gene family, prp1-1, is activated at early stages of potato infection with the late blight fungus Phytophthora infestans. In this paper we present evidence that mRNA encoded by prp1-1 does not accumulate in response to abiotic environmental cues which stimulate transcription of other defence-related genes. Regulatory elements were identified in the 5' terminal region of prp1-1 by assaying the expression pattern of chimeric promoter/beta-glucuronidase gene constructs in transgenic potato. A 273 bp fragment comprising the promoter sequence between positions -402 and -130 was sufficient for rapid and strictly localized transcriptional activation at infection sites during the development of late blight disease. Like the native promoter, this truncated promoter did not mediate transcriptional activation in response to other abiotic stimuli. The use of the identified regulatory region to generate conditional mutations selectively at infection sites is discussed.
gene encoding heat-shock Brmmiu /actucae
- H S Judelson
- Michelmore
Judelson, H.S. and Michelmore, gene encoding heat-shock Brmmiu /actucae. Gene 79 ( 1989) 207-2 17