[show abstract][hide abstract] ABSTRACT: BACKGROUND: Anthracnose of lentil, caused by the hemibiotrophic fungal pathogen Colletotrichum truncatum is a serious threat to lentil production in western Canada. Colletotrichum truncatum employs a bi-phasic infection strategy characterized by initial symptomless biotrophic and subsequent destructive necrotrophic colonization of its host. The transition from biotrophy to necrotrophy (known as the biotrophy-necrotrophy switch [BNS]) is critical in anthracnose development. Understanding plant responses during the BNS is the key to designing a strategy for incorporating resistance against hemibiotrophic pathogens either via introgression of resistance genes or quantitative trait loci contributing to host defense into elite cultivars, or via incorporation of resistance by biotechnological means. RESULTS: The in planta BNS of C. truncatum was determined by histochemical analysis of infected lentil leaf tissues in time-course experiments. A total of 2852 lentil expressed sequence tags (ESTs) derived from C. truncatum-infected leaf tissues were analyzed to catalogue defense related genes. These ESTs could be assembled into 1682 unigenes. Of these, 101 unigenes encoded membrane and transport associated proteins, 159 encoded proteins implicated in signal transduction and 387 were predicted to be stress and defense related proteins (GenBank accessions: JG293480 to JG293479). The most abundant class of defense related proteins contained pathogenesis related proteins (encoded by 125 ESTs) followed by heat shock proteins, glutathione S-transferase, protein kinases, protein phosphatase, zinc finger proteins, peroxidase, GTP binding proteins, resistance proteins and syringolide-induced proteins. Quantitative RT-PCR was conducted to compare the expression of two resistance genes of the NBS-LRR class in susceptible and partially resistant genotypes. One (contig186) was induced 6 days post-inoculation (dpi) in a susceptible host genotype (Eston) whereas the mRNA level of another ( LT21-1990) peaked 4 dpi, suggesting roles in conditioning the susceptibility and conferring tolerance to the pathogen, respectively. CONCLUSIONS: Data obtained in this study suggest that lentil cells recognize C. truncatum at the BNS and in response, mount an inducible defense as evident by a high number of transcripts (23% of the total pathogen-responsive lentil transcriptome) encoding defense related proteins. Temporal expression polymorphism of defense related genes could be used to distinguish the response of a lentil genotype as susceptible or resistant.
[show abstract][hide abstract] ABSTRACT: The hemibiotrophic fungus Colletotrichum truncatum causes anthracnose disease on lentil, and few other grain legumes. It shows an initial symptomless intracellular growth wherein colonized host cells remain viable (biotrophy) and then switches to necrotrophic growth killing the colonized host plant tissues. We herein report a novel effector gene CtNUDIX from C. truncatum that exclusively expresses during the late-biotrophic phase (before the switch to necrotrophy) and elicits a hypersensitive response (HR)-like cell death in tobacco leaves transiently expressing the effector. CtNUDIX homologs, which contain a signal peptide and a Nudix hydrolase domain, may be unique to hemibiotrophic fungal and fungus-like plant pathogens. CtNUDIX lacking a signal peptide or a Nudix motif failed to induce cell death in tobacco. Expression of CtNUDIX:eGFP in tobacco suggested that the fusion protein might act on the host cell plasma membrane. Overexpression of CtNUDIX in C. truncatum and the rice blast pathogen, Magnaporthe oryzae resulted in incompatibility with hosts lentil and barley, respectively, by causing a HR-like response in infected host cells associated with the biotrophic invasive hyphae. These results suggest that C. truncatum and possibly M. oryzae elicits cell death to signal the transition from biotrophy to necrotrophy.
[show abstract][hide abstract] ABSTRACT: The rice blast pathogen, Magnaporthe oryzae has been widely used as a model pathogen to study plant infection-related fungal morphogenesis, such as penetration via appressorium and plant-microbe interactions at the molecular level. Previously, we identified a gene encoding peroxisomal alanine: glyoxylate aminotransferase 1 (AGT1) in M. oryzae and demonstrated that the AGT1 was indispensable for pathogenicity. The AGT1 knockout mutants were unable to penetrate the host plants, such as rice and barley, and therefore were non-pathogenic. The inability of ∆Moagt1 mutants to penetrate the susceptible plants was likely due to the disruption in coordination of the β-oxidation and the glyoxylate cycle likely resulted from a blockage in lipid droplet mobilization and eventually utilization during conidial germination and appressorium morphogenesis, respectively. Here, we further demonstrate the role of AGT1 in lipid mobilization by in vitro germination assays and confocal microscopy.
[show abstract][hide abstract] ABSTRACT: Protein extraction is a critical step in any proteomics study. Since most fungi possess a robust cell wall, efficient isolation
of total proteins has become challenge to fungal proteomics. To circumvent this bottleneck of fungal proteomics, we standardized
a protocol named as Mg/CHAPS extraction by comparing with an established method of protein extraction (Tris/EDTA extraction),
using 2-DE and MALDI-TOF MS. Total mycelial proteins were isolated using both protocols from Magnaporthe grisea (causal agent of rice blast disease). Six hundred forty two proteins were resolved on two 2-DE gels corresponding to mycelial
proteomes isolated by Mg/CHAPS and Tris/EDTA. Mycelial proteome extracted by Mg/CHAPS showed higher number protein spots than
to Tris/EDTA. Quantitative analysis of mycelial proteome, histogram and MS analyses of a protein spot suggested that Mg/CHAPS
extraction is more effective than the widely used protocol i.e. Tris/EDTA.
KeywordsFungal proteomics-Mg/CHAPS extraction-Tris-EDTA extraction-Protein extraction techniques-Mycelial proteome
Indian Journal of Microbiology 04/2012; 50:127-131. · 0.46 Impact Factor
[show abstract][hide abstract] ABSTRACT: Proteomics, the systematic analysis of the proteome, is a powerful tool in the post-genomic era. Proteomics studies have examined
global changes in proteomes of phytopathogenic fungi, oomycetes and their hosts during compatible or incompatible interactions.
This article compiles proteomics reports in order to decipher the molecular mechanisms underlying fungal development (infection-related
morphogenesis), fungal or oomycete—host plant interactions, and phytopathogenesis.
European Journal of Plant Pathology 04/2012; 126(1):81-95. · 1.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: The role of β-oxidation and the glyoxylate cycle in fungal pathogenesis is well documented. However, an ambiguity still remains over their interaction in peroxisomes to facilitate fungal pathogenicity and virulence. In this report, we characterize a gene encoding an alanine, glyoxylate aminotransferase 1 (AGT1) in Magnaporthe oryzae, the causative agent of rice blast disease, and demonstrate that AGT1 is required for pathogenicity of M. oryzae. Targeted deletion of AGT1 resulted in the failure of penetration via appressoria; therefore, mutants lacking the gene were unable to induce blast symptoms on the hosts rice and barley. This penetration failure may be associated with a disruption in lipid mobilization during conidial germination as turgor generation in the appressorium requires mobilization of lipid reserves from the conidium. Analysis of enhanced green fluorescent protein expression using the transcriptional and translational fusion with the AGT1 promoter and open reading frame, respectively, revealed that AGT1 expressed constitutively in all in vitro grown cell types and during in planta colonization, and localized in peroxisomes. Peroxisomal localization was further confirmed by colocalization with red fluorescent protein fused with the peroxisomal targeting signal 1. Surprisingly, conidia produced by the Δagt1 mutant were unable to form appressoria on artificial inductive surfaces, even after prolonged incubation. When supplemented with nicotinamide adenine dinucleotide (NAD(+))+pyruvate, appressorium formation was restored on an artificial inductive surface. Taken together, our data indicate that AGT1-dependent pyruvate formation by transferring an amino group of alanine to glyoxylate, an intermediate of the glyoxylate cycle is required for lipid mobilization and utilization. This pyruvate can be converted to non-fermentable carbon sources, which may require reoxidation of NADH generated by the β-oxidation of fatty acids to NAD(+) in peroxisomes. Therefore, it may provide a means to maintain redox homeostasis in appressoria.
PLoS ONE 01/2012; 7(4):e36266. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Hemibiotrophic phytopathogenic fungi cause devastating diseases in agronomically important crops. These fungal pathogens exploit a stealth bi-phasic infection strategy to colonize host plants. Their morphological and nutritional transition from biotrophy (characterized by voluminous intracellular primary hyphae) to necrotrophy (characterized by thin secondary hyphae) known as the biotrophy-necrotrophy switch (hemibiotrophy) is critical in symptom and disease development. To establish successful hemibiotrophic parasitism, pathogens likely secrete suites of proteins at the switch that constitute the biotrophy-necrotrophy switch secretome. To catalogue such proteins, a directional cDNA library was constructed from mRNA isolated from infected Lens culinaris leaflet tissues displaying the switch of Colletotrichum truncatum, and 5000 expressed sequence tags (ESTs) were generated. Four potential groups (hydrolytic enzymes, cell envelope-associated proteins [CEAPs], candidate effectors and proteins with diverse functions) were identified from pathogen-derived ESTs. Expression profiling of transcripts encoding CEAPs and candidate effectors in an infection time-course revealed that the majority of these transcripts were expressed or induced during the necrotrophic phase and repressed during the biotrophic phase of in planta colonization, indicating the massive accumulation of proteins at the switch. Taken together, our data suggest that the hemibiotrophic mode of fungal proliferation entails complex interactions of a pathogen with its host wherein the pathogen requires live host cells prior to switching to the necrotrophic phase. The microbial proteins employed during pathogenesis are likely to have defined roles at specific stages of pathogenesis.
[show abstract][hide abstract] ABSTRACT: Colletotrichum truncatum is a haploid, hemibiotrophic, ascomycete fungal pathogen that causes anthracnose disease on many economically important leguminous crops. This pathogen exploits sequential biotrophic- and necrotrophic- infection strategies to colonize the host. Transition from biotrophy to a destructive necrotrophic phase called the biotrophy-necrotrophy switch is critical in symptom development. C. truncatum likely secretes an arsenal of proteins that are implicated in maintaining a compatible interaction with its host. Some of them might be transition specific.
A directional cDNA library was constructed from mRNA isolated from infected Lens culinaris leaflet tissues displaying the biotrophy-necrotrophy switch of C. truncatum and 5000 expressed sequence tags (ESTs) with an average read of > 600 bp from the 5-prime end were generated. Nearly 39% of the ESTs were predicted to encode proteins of fungal origin and among these, 162 ESTs were predicted to contain N-terminal signal peptides (SPs) in their deduced open reading frames (ORFs). The 162 sequences could be assembled into 122 tentative unigenes comprising 32 contigs and 90 singletons. Sequence analyses of unigenes revealed four potential groups: hydrolases, cell envelope associated proteins (CEAPs), candidate effectors and other proteins. Eleven candidate effector genes were identified based on features common to characterized fungal effectors, i.e. they encode small, soluble (lack of transmembrane domain), cysteine-rich proteins with a putative SP. For a selected subset of CEAPs and candidate effectors, semiquantitative RT-PCR showed that these transcripts were either expressed constitutively in both in vitro and in planta or induced during plant infection. Using potato virus X (PVX) based transient expression assays, we showed that one of the candidate effectors, i. e. contig 8 that encodes a cerato-platanin (CP) domain containing protein, unlike CP proteins from other fungal pathogens was unable to elicit a hypersensitive response (HR).
The current study catalogues proteins putatively secreted at the in planta biotrophy-necrotrophy transition of C. truncatum. Some of these proteins may have a role in establishing compatible interaction with the host plant.
[show abstract][hide abstract] ABSTRACT: Triglyceride lipases catalyze the reversible degradation of glycerol esters with long-chain fatty acids into fatty acids and glycerol. In silico analysis of 5'-end flanking sequence of the gene LIP1 encoding a triglyceride lipase from the wheat head blight pathogen Fusarium graminearum revealed the presence of several cis-regulatory elements. To delineate the function of these regulatory elements, we constructed a series of deletion mutants in the LIP1 promoter region fused to the open reading frame of a green fluorescent protein (GFP) and assayed the promoter activity. Analysis of GFP expression levels in mutants indicated that a 563-bp promoter sequence was sufficient to drive the expression of LIP1 and regulatory elements responsible for the gene induction were located within the 563-372bp region. To further investigate the regulatory elements, putative cis-acting elements spanned within the 563-372bp region were mutated using a targeted mutagenesis approach. A CCAAT box, a CreA binding site, and a fatty acid responsive element (FARE) were identified and confirmed to be required for the basal expression of LIP1, glucose suppression and fatty acid induction, respectively.
Microbiological Research 02/2011; 166(8):618-28. · 1.99 Impact Factor
[show abstract][hide abstract] ABSTRACT: The rice blast disease caused by Magnaporthe oryzae is a major constraint on world rice production. The conidia produced by this fungal pathogen are the main source of disease dissemination. The morphology of conidia may be a critical factor in the spore dispersal and virulence of M. oryzae in the field. Deletion of a conidial morphology regulating gene encoding putative transcriptional regulator COM1 in M. oryzae resulted in aberrant conidial shape, reduced conidiation and attenuated virulence.
In this study, a two-dimensional gel electrophoresis/matrix assisted laser desorption ionization- time of flight mass spectrometry (2-DE/MALDI-TOF MS) based proteomics approach was employed to identify the cellular and molecular components regulated by the COM1 protein (COM1p) that might contribute to the aberrant phenotypes in M. oryzae. By comparing the conidial proteomes of COM1 deletion mutant and its isogenic wild-type strain P131, we identified a potpourri of 31 proteins that exhibited statistically significant alterations in their abundance levels. Of these differentially regulated proteins, the abundance levels of nine proteins were elevated and twelve were reduced in the Δcom1 mutant. Three proteins were detected only in the Δcom1 conidial proteome, whereas seven proteins were apparently undetectable. The data obtained in the study suggest that the COM1p plays a key role in transcriptional reprogramming of genes implicated in melanin biosynthesis, carbon and energy metabolism, structural organization of cell, lipid metabolism, amino acid metabolism, etc. Semi-quantitative RT-PCR analysis revealed the down-regulation of genes encoding enzymes involved in melanin biosynthesis in the COM1 mutant.
Our results suggest that the COM1p may regulate the transcription of genes involved in various cellular processes indispensable for conidial development and appressorial penetration. These functions are likely to contribute to the effects of COM1p upon the aberrant phenotypes of M. oryzae.
Biology Direct 11/2010; 5:61. · 2.72 Impact Factor
[show abstract][hide abstract] ABSTRACT: Since genomes of rice and blast fungus are sequenced, global profiling of gene expression i.e. transcriptomics has become an important tool to decipher molecular mechanisms underlying rice-blast interaction and infection related morphogenesis (appressorium formation and maturation, formation of penetration peg). This article summarises transcriptomics studies conducted on rice blast fungus.
Archives of Phytopathology and Plant Protection 04/2010; 43(8):723-727.
[show abstract][hide abstract] ABSTRACT: The rice blast fungus genome has been sequenced. Now the major task is to monitor the global changes in its proteome for dissecting the molecular mechanisms underlying rice-blast interaction, fungal development (conidial germination, appressorium morphogenesis and formation of penetration peg), pathogenesis. Proteome analysis of rice blast fungus can be instrumental in elucidating these mechanisms at protein level. This article summarises proteomics studies conducted on rice blast fungus and its interaction with rice.
Archives of Phytopathology and Plant Protection 01/2010; 43(2):149-153.
[show abstract][hide abstract] ABSTRACT: Sequencing of over 40 fungal and oomycete genomes has been completed. The next major challenge in modern fungal/oomycete biology is now to translate this plethora of genome sequence information into biological functions. Reverse genetics has emerged as a seminal tool for functional genomics investigations. Techniques utilized for reverse genetics like targeted gene disruption/replacement, gene silencing, insertional mutagenesis, and targeting induced local lesions in genomes will contribute greatly to the understanding of gene function of fungal and oomycete pathogens. This paper provides an overview on high-throughput reverse genetics approaches to decode fungal/oomycete genomes.
Comparative and Functional Genomics 01/2009; · 0.92 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have now entered in the post-genomic era, where we have knowledge of plethora of fungal genomes and cutting edge technology is available to study global mRNA, protein and metabolite profiles. These so-called 'omic' technologies (transcriptomics, proteomics and metabolomics) provide the possibility to characterize plant-pathogen interactions and pathogenesis at molecular level. This article provides an overview of transcriptomics and its applications in fungal plant pathology.
Microbiological Research 02/2007; 162(4):285-98. · 1.99 Impact Factor
[show abstract][hide abstract] ABSTRACT: Proteomics, the global analysis of proteins, will contribute greatly to our understanding of gene function in the post-genomic era. This review summarizes recent developments in fungal proteomics and also generalizes protocols for sample preparation from plant pathogenic fungi. Challenges and future perspectives of proteomics are discussed as well.
Microbiological Research 02/2007; 162(3):193-200. · 1.99 Impact Factor