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Fungal phytopathogens encode functional homologues of plant rapid alkalinisation factor (RALF) peptides

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In this paper we describe the presence of genes encoding close homologues of an endogenous plant peptide, rapid alkalinisation factor (RALF), within the genomes of 26 species of phytopathogenic fungi. Members of the RALF family are key growth factors in plants, and the sequence of the RALF active region is well conserved between the plant and fungal proteins. RALF1-like sequences were observed in most cases; however, RALF27-like sequences were present in the Sphaerulina musiva and Septoria populicola genomes. These two species are pathogens of poplar and interestingly, the closest relative to their respective RALF genes is a poplar RALF27-like sequence. RALF peptides control cellular expansion during plant development, but were originally defined based on their ability to induce rapid alkalinisation in tobacco cell cultures. To test whether the fungal RALF peptides were biologically active in plants, we synthesized RALF peptides corresponding to those encoded by two sequenced genomes of the tomato pathogen Fusarium oxysporum f. sp. lycopersici. One of these peptides inhibited the growth of tomato seedlings and elicited responses in tomato and Nicotiana benthamiana typical of endogenous plant RALF peptides (ROS burst, induced alkalinisation and MAP kinas activation). Gene expression analysis confirmed that a RALF-encoding gene in Fusarium oxysporum f. sp. lycopersici was expressed during infection on tomato. However a subsequent reverse genetics approach revealed that the RALF peptide was not required by Fusarium oxysporum f. sp. lycopersici for infection on tomato roots. This study has demonstrated the presence of functionally active RALF peptides encoded within phytopathogens that harbour an as yet undetermined role in the plant-pathogen interactions. This article is protected by copyright. All rights reserved.
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Fungal phytopathogens encode functional homologues of
plant rapid alkalinisation factor (RALF) peptides
ELISHA THYNNE1, ISABEL M. L. SAUR1, JAIME SIMBAQUEBA1, HUW A.
OGILVIE2,3, YVONNE GONZALEZ-CENDALES1, OLIVER MEAD1, ADAM
TARANTO1, ANN-MAREE CATANZARITI1, MEGAN C. MCDONALD1,
BENJAMIN SCHWESSINGER1, DAVID A. JONES1*, JOHN P. RATHJEN1* AND
PETER S. SOLOMON1*
1Plant Sciences Division, 2Evolution, Ecology and Genetics Division, Research School of
Biology, The Australian National University, Canberra, Australia, 2601
3Computational Evolution Group, The University of Auckland, Auckland, New Zealand
Key words: Rapid Alkalinisation Factor, RALF, Phytopathogen Effectors, Fusarium
* Correspondence: Email: peter.solomon@anu.edu.au, john.rathjen@anu.edu.au,
david.jones@anu.edu.au
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as an
‘Accepted Article’, doi: 10.1111/mpp.12444
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SUMMARY
In this paper we describe the presence of genes encoding close homologues of an
endogenous plant peptide, rapid alkalinisation factor (RALF), within the genomes of 26
species of phytopathogenic fungi. Members of the RALF family are key growth factors in
plants, and the sequence of the RALF active region is well conserved between the plant
and fungal proteins. RALF1-like sequences were observed in most cases; however,
RALF27-like sequences were present in the Sphaerulina musiva and Septoria populicola
genomes. These two species are pathogens of poplar and interestingly, the closest
relative to their respective RALF genes is a poplar RALF27-like sequence. RALF peptides
control cellular expansion during plant development, but were originally defined based
on their ability to induce rapid alkalinisation in tobacco cell cultures. To test whether
the fungal RALF peptides were biologically active in plants, we synthesized RALF
peptides corresponding to those encoded by two sequenced genomes of the tomato
pathogen Fusarium oxysporum f. sp. lycopersici. One of these peptides inhibited the
growth of tomato seedlings and elicited responses in tomato and Nicotiana benthamiana
typical of endogenous plant RALF peptides (ROS burst, induced alkalinisation and MAP
kinas activation). Gene expression analysis confirmed that a RALF-encoding gene in
Fusarium oxysporum f. sp. lycopersici was expressed during infection on tomato.
However a subsequent reverse genetics approach revealed that the RALF peptide was
not required by Fusarium oxysporum f. sp. lycopersici for infection on tomato roots. This
study has demonstrated the presence of functionally active RALF peptides encoded
within phytopathogens that harbour an as yet undetermined role in the plant-pathogen
interactions.
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INTRODUCTION
Fungal phytopathogens are a major threat to all plant ecosystems, both natural and
agricultural (Stukenbrock & McDonald, 2008). Therefore it is crucial to understand how
fungal phytopathogens have co-evolved with their plant hosts to enable better
management of crop losses and preserve natural biodiversity. The mechanisms by
which pathogenic fungi manipulate their plant hosts are complex and diverse. It is now
recognised that pathogen effector molecules (proteins or secondary metabolites) have a
significant role in manipulating host plants to facilitate infection (de Jonge et al., 2011).
For example, biotrophic fungi produce effectors that allow the fungus to elude or
manipulate host defences. Conversely, necrotrophic fungi produce effectors that induce
host cell necrosis thereby producing dead plant tissue for the fungus to colonise. Some
effectors are vital for host infection; others are not strictly necessary but assist in fully
exploiting host resources (De Wit et al., 2009). Most fungal effector genes remain
undiscovered, but the increasing availability of fully sequenced fungal genomes has
enabled new methods for the identification of putative effector genes using comparative
genomics and candidate gene prediction tools.
The advent of next-generation sequencing (NGS) has dramatically increased the
number of whole genome sequences available for organisms from all kingdoms of life.
Access to these data has allowed researchers to compare genes from disparate
organisms (Thynne et al., 2015a) and provided significant insights into the genes
involved in phytopathogen emergence and niche specialization (Klosterman et al., 2011,
Gardiner et al., 2012) including the identification of pathogen genes related to host
genes, either through horizontal gene transfer (HGT) or convergent evolution. By
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studying the molecular evolution of these genes, we can develop hypotheses about their
function, including their potential roles as effectors.
Horizontal transfer of pathogenicity genes between fungi is a well-known
phenomenon and an active area of research (Richards et al., 2011, Friesen et al., 2006,
Gardiner et al., 2012, Thynne et al., 2015a, Klosterman et al., 2011), although the exact
mechanisms of genetic transfer are poorly understood. Recent studies have also
demonstrated a role for plant-to-microorganism gene transfer leading to increased
virulence. For example, the secreted proteinaceous Verticillium effector Ave1, which
promotes pathogen virulence on the plant host, is hypothesized to have a plant origin
(de Jonge et al., 2012). Other examples exist where plant-interacting organisms have
apparently acquired plant growth and cell structure altering factors. For example, C-
terminally encoded peptides (CEP) were identified in the genome assemblies of
phytopathogenic root knot nematodes (Bobay et al., 2013). Similarly, homologues of
plant expansins were identified in the genomes of a range of bacteria and fungi
(Nikolaidis et al., 2014). CEPs and expansins are growth regulatory and plant-cell
loosening molecules, respectively (Cosgrove, 2000, Delay et al., 2013). These molecules
have been postulated to assist in plant colonization, inhabitation, and/or parasitisation
(Soanes & Richards, 2014). Pathogen genes of plant origin are therefore promising
targets for further study as potential virulence factors.
The plant rapid alkalinisation factor (RALF) genes encode secreted peptides that
were first identified through their ability to trigger a rapid increase in extracellular pH
when added to plant cell suspensions (Pearce et al., 2001). RALF peptides have roles in
the development and regulation of plant roots, root hairs, legume nodules and pollen
tubes (Pearce et al., 2010, Murphy & De Smet, 2014). Subsequent in silico analyses of the
Arabidopsis thaliana genome have identified 34 genes within the RALF family (Olsen et
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al., 2002). These genes were named RALF1 to RALF34, and all homologues since
identified in other species have been named according to their similarities to members
of the A. thaliana family. Functional studies on a tobacco RALF peptide revealed that
proteolytic cleavage of a larger precursor protein generates a 49 amino acid active
peptide (Fig. 1) (Srivastava et al., 2009). Mutational analysis has identified several
motifs essential for RALF activity including a YISY motif and four conserved cysteine
residues that are required for disulphide bonding (Pearce et al., 2010). Recently the
Arabidopsis receptor for RALF1 was identified as the plasma membrane receptor kinase
FERONIA. Activation of FERONIA results in phosphorylation of plasma membrane H+
ATPase 2 at Ser-899, causing inhibition of proton transport (Haruta et al., 2014).
In this study, we describe the distribution of genes encoding RALF homologues
amongst a diverse range of plant pathogenic fungal species and RALF-like hybrid genes
in a limited number of bacterial species. Functional testing of synthetic versions of two
fungal RALF peptides showed that plants perceive and respond to the fungal peptides.
These data suggest that these fungal RALF genes enable pathogenic fungi to manipulate
host plant physiology.
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RESULTS
Distribution and variation of RALF peptide sequences in the fungal and bacterial
kingdoms
The search programs BLASTp and tBLASTn were used to screen publically available
fungal genomes for homologues of plant RALF proteins using the full-length protein
sequences of Arabidopsis thaliana plant RALF genes 1 through 34 (Table S1). Using this
approach, 26 different species of fungi were found to possess genes encoding the
processed RALF peptide domain (Fig. 2, Table 1). These include the class
Pucciniomycetes of the Basidiomycota and the classes Dothideomycetes and
Sordariomycetes of the Ascomycota. All of the fungi found to possess RALF homologues
are plant pathogens. Both biotrophic and necrotrophic fungi are represented in this list.
Phylogenetic analysis of plant and fungal RALF peptide sequences showed that the
fungal RALF homologues are interspersed amongst the plant RALFs (Fig. S1, see
Supporting Information). Many of the fungal homologues resolved with the plant RALF1
proteins (Fig. S1, see Supporting Information). The Pseudocercospora fijiensis RALF
homologue clustered with RALF homologues of Musa acuminate malaccensis. However,
this is only supported with a low posterior probability of 33%. The Septoria populicola
and Sphaerulina musiva RALF homologues clustered with the plant RALF27-like
proteins with 100% posterior support (Fig. 2, Fig. S1, see Supporting Information). The
best BLASTp hit to a complete plant protein for both fungal sequences was a RALF27-
like protein from Populus trichocarpa (E-values 3e-14 and 4e-13), however this is only
weakly supported by the gene tree analysis, with a posterior probability of 50% (Fig. 2,
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Fig. S1, see Supporting Information). Note that there was no detectable homology
between the N-terminal pro-peptide domains of the plant and fungal RALF sequences
Analysis of the Fusarium species showed evidence of four divergent groups of RALF
homologues (Fig. 3, Table 2), which we designated Groups I to IV. Members of Group I
are present in all isolates of F. oxysporum (Fox) that have been sequenced to date,
although the Group I RALF genes of two Fox isolates encode sequence variations
suggesting loss of function (a premature stop codon in the human pathology isolate
FOSC 3-a, and a tyrosine in place of a conserved cysteine in the non-pathogen Fo47).
Group I can be subdivided into two subgroups A and B, with subform IB also present in
F. verticillioides, F. fujikuroi and F. circinata (Fig. 3). Interestingly, subform IA
distinguishes two tomato pathogens, Fox f. sp. lycopersici (Fol) isolate MN25 and Fox f.
sp. radicis-lycopersici (Forl) from a third, Fol isolate 4287. The divergent Fol
homologues, here designated RALF-B (from 4287) and RALF-C (from MN25), were
chosen for further study.
The Group II family of RALF homologues has only two members, one from Fox FOSC
3-a, which could potentially complement the aberrant FOSC 3-a Group I homologue and
the other from Fox f. sp. melonis, which contains an apparent frame-shift mutation (Fig.
3). The Group III family of RALF homologues has only three members, one from Forl
CL57 and the others from F. graminearum and F. pseudograminearum (Fig. 3).
Genes encoding a fourth family of RALF-like proteins (Group IV) were also found in
Fusarium. These differ from the three families described above by a central deletion
encompassing the conserved cysteines that form the first disulphide bond (Fig. 3).
Nevertheless, the sequence conservation within this family suggests the retention of
function, but whether they possess the same function as the fungal RALF homologues
described above requires further investigation. This gene family can also be divided into
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two sequence-divergent subgroups that seem to be distributed in a mutually exclusive
fashion between different formae speciales of Fox, although two (vasinfectum and Fol
4287) do not possess either form. Interestingly, Group IV genes seem to be absent from
plants and other fungal genomes, suggesting that they are unique to Fusarium.
Notably, all four possible combinations of the Group I and IV RALF variants occur in
Fox, indicating a polyphyletic origin for at least some of these gene combinations. This is
a curious finding given that these genes also occur in F. verticillioides, F. fujikuroi and F.
circinata, suggesting that they are located on the core conserved set of non-mobile
chromosomes in Fusarium rather than the mobile lineage-specific chromosomes that
seem to determine host specificity in Fox (Ma et al. 2010). Indeed, the RALF-B gene is
encoded by core chromosome 12 in Fol 4287.
Subsequent to the preliminary database search, we annotated a number of new RALF
sequences within the genomes of members of the Botryosphaeriaceae. Similarly to the
Fusarium spp., members of the Botryosphaeriaceae family showed evidence of their own
three divergent groups of RALF homologues (Table 3, Fig. S2, see Supporting
Information). These three groups were designated BI-BIII. Amino acid variations
between these three divergent groups are highlighted in Fig. S2A (see Supporting
Information). BI contains an amino acid string (“ISNGAM”) apparently rare among both
fungal RALFs (only non-Botryosphaeriaceae RALF is in C. higginsianum) and plant RALFs
(only observed in Brachypodium distachyon). BII is similar in amino acid composition to
the Fusarium group I. BIII is similar, and perhaps divergent from BII. Botryosphaeria
dothidea contains two RALF homologues (BI and BII). B. dothidea’s BI RALF homologue
was previously un-annotated. Diplodia seriata contains one RALF homologue (BIII).
Neofusicoccum parvum contains one (BI). M. phaseolina contains four RALF homologues
(BI, two BIIs, and BIII). All but one of M. phaseolina’s RALFs were previously un-
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annotated. With four predicted RALF peptides, M. phaseolina has the highest number of
predicted fungal RALF peptides identified in this study. One of the BII RALF-peptides
shares a conserved RALF peptide-domain with the mature RALF peptide from soybean
specie (Glycine max and Glycine soja). For this region these species have a 100% amino
acid identity. Variation among these species is found in the N-terminal and C-terminal of
these regions. Additionally, common bean (Phaseolus vulgaris) has a 98% sequence
similarity for this conserved region and mung bean (Vigna radiate), has a 100%
sequence similarity for 97% of the query coverage. The only species of
Botryosphaeriaceae analysed in this study not to contain RALF peptides are three closely
related species, Eutiarosporella darliae, E. pseudodarliae, and E. tritici-australis. These
are the causal agents of white grain disorder (WGD) in wheat (Thynne et al. 2015b).
We re-performed the database searches using BLASTp, this time searching the
bacterial portal of the NCBI database. We identified certain members of Actinobacteria
that possess putative secreted proteins with an incorporated RALF peptide domain
motif (Fig. S3, see Supporting Information). In both fungal and plant RALFs, this
particular motif is located at the C-terminal of the mature peptide. Similarly, in these
bacterial proteins, the RALF domain motif is at the C-terminal of the protein. Unlike
plant and fungal RALFs, the bacterial RALF-domain-containing proteins also contain a
domain homologous to the S1 pertussis toxin subunit. These bacterial species include a
number of plant-pathogenic species, for example Streptomyces acidiscabies (a pathogen
of potatoes) (Huguet-Tapia et al., 2012).
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Tomato seedling growth and development is severely inhibited by a synthetic
RALF-B peptide
Endogenous RALF peptides arrest the growth and development of plant roots (Pearce et
al., 2010). To determine if fungal RALF peptides share this property, we synthesized
RALF peptides based on RALF2 of tomato (Sl-RALF, NCBI GenBank GI:460366641), and
those of Fol race 2 isolate 4287 and race 3 isolate MN25 (RALF-B and RALF-C
respectively) (Fig. 2, Fig. S4, see Supporting Information). Fol 4287 was the first fully
sequenced F. oxysporum isolate, and MN25 is a representative of race 3, which appeared
in the 1980s and overcame resistance to races 1 and 2 (Bournival et al., 1989). A non-
functional mutant of Sl-RALF lacking the required YISY motif (Sl-RALF) was used as a
negative control. Germinating tomato seeds were grown in media containing each RALF
peptide at 10 μM (Fig. 4), or with no added peptide. Untreated seedlings remained
healthy and showed normal development of roots and shoots. Seedlings grown in media
containing Sl-RALF developed less biomass than the untreated controls, but this was not
significantly different to the Sl-RALF treatment (Fig. 4A). However, the Sl-RALF treated
seedlings were developmentally stunted with minimal root-formation compared to the
Sl-RALF-treated seedlings (Fig. 4B). The growth and root development of seedlings
treated with synthetic RALF-B peptide was severely inhibited relative to mock- and Sl-
RALF-treated seedlings. This was reflected in their significantly lower average weights.
In contrast, the synthetic RALF-C peptide did not inhibit root growth. There was no
significant difference in average biomass between seedlings treated with RALF-C and
the mock and Sl-RALF treatments. Similarly, the seedlings appeared healthy, with well-
developed root systems.
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The RALF-B peptide elicits recognised plant RALF responses
The receptor for the Arabidopsis RALF1 peptide is the CrRLK1L family receptor
FERONIA (FER) (Haruta et al., 2014). As FER function is associated with accumulation of
extracellular ROS (Wolf & Höfte, 2014), and this is also a typical consequence of plant
defence activation, we asked if synthetic Fol-RALF peptides could also elicit ROS
production. Treatment of leaf discs of tomato and another Solanaceous species,
Nicotiana benthamiana, with Sl-RALF at 10 μM induced a ROS burst. The ROS burst
elicited by Sl-RALF was stronger in tomato than N. benthamiana. In contrast, RALF-B
(but not RALF-C) induced a massive ROS burst in both solanaceous species, only
minutes after treatment. RALF-C was not active in the assays. Neither the mock nor the
Sl-RALF treatments elicited a significant ROS response in leaves of either species’.
The dose response for fungal RALF peptides was also determined. As the ROS bursts
induced by RALF-B were comparable between tomato and N. benthamiana, the activity
of this peptide was tested in N. benthamiana. RALF-B was active down to a
concentration of 10 nM, with progressive increases in ROS output observed up to 500
nM (Fig. S5, see Supporting Information). In contrast, ROS output subsequent to
increasing concentrations of RALF-C did not exceed control levels providing further
evidence that the peptide does not elicit a typical RALF response in plants.
The ability of RALF-B to induce alkalinisation was also determined. RALF peptides
were originally named as such due to their strong and rapid alkalinisation of tobacco
suspension-cultured cells (Pearce et al., 2001). To test the ability of RALF-B to induce
alkalinisation, the peptide was incubated with cut leaf strips from both tomato and N.
benthamiana. For both plants, RALF-B elicited an alkalinisation response not
significantly different to that of the tomato RALF peptide (Fig. 5C-D).
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Similarly, the activation of mitogen-activated protein kinases (MAPKs), a known
RALF response (Pearce et al., 2001), was also investigated. Peptide induced MAPK
activation was measured in N. benthamiana leaf discs as confirmation of the variation of
activity in response to the different peptides. Treatments with 10 µM Sl-RALF or RALF-
C activated MAPK progressively, with greater activity detected at 15 min after peptide
addition (Fig. S6, see Supporting Information). Interestingly, addition of only 100 nM
RALF-B (i.e. 100x times less than Sl-RALF) caused strong activation of MAPK that
peaked at 5 min and showed decline by 15 min. This mirrors the ROS profile arising
from RALF-B treatment. Overall, these data (ROS burst, alkalinisation and MAPK
activation) prove that plant cells perceive fungal RALF peptides.
The Fusarium oxysporum f. sp. lycopersici RALF-B gene is expressed during
infection of tomato roots
If RALF-B plays a role in fungal pathogenicity, it should be expressed during infection of
tomato roots by Fol isolates carrying the RALF-B gene. A reverse transcriptase (RT) PCR
analysis was carried out on roots of the susceptible tomato cultivar M82 infected with
Fol isolate #1943, which carries the RALF-B gene, at 3 and 6 days post inoculation (dpi).
No RALF-B expression was detected in samples from roots of mock-inoculated tomato
plants or five-day-old mycelia grown in vitro, but expression was detected in the 3 and 6
dpi samples (Fig. 6). The FEM1 gene (Schoffelmeer et al., 2001) was used as a positive
control for fungal gene expression. The RALF-B primers flanked an intron, thus yielding
different-sized products for cDNA and genomic DNA (Fig. 6). No RALF-B or FEM1
genomic-DNA products were detected in cDNA samples (Fig. 6) and no RALF-B or FEM1
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products were detected in samples prepared without reverse transcriptase (not shown),
indicating that the RALF-B and FEM1 bands observed at 3 and 6 dpi are authentic RT-
PCR products. The absence of RALF-B expression in mycelia grown in vitro suggests that
RALF-B expression is induced during plant infection, consistent with a potential role as
an effector.
Effect of knocking out the RALF-B gene in Fol race 3
To test the role of the RALF-B gene in Fol pathogenicity, knockout (ΔRALF) mutants
were obtained by Agrobacterium-mediated transformation, using a binary T-DNA vector
carrying a hygromycin resistance gene as a selectable marker for gene replacement and
a thymidine kinase gene as a counter-selectable marker against ectopic T-DNA
insertions (Fig. S7A, see Supporting Information). A total of 44 Fol transformants that
showed resistance to hygromycin and 5'-fluoro-2'-deoxyuridine were screened by PCR,
confirming deletion of the RALF coding sequence in four transformants (named Δ2, Δ23,
Δ24 and Δ31) (Fig. S7B, see Supporting Information).
The four Fol-ΔRALF transformants were then used to evaluate the effect of loss of the
RALF gene on virulence. The roots of three-week-old plants of the tomato cultivar M82
(susceptible to Fol race 3) were wounded and subsequently inoculated with Fol race 3, a
transformant with an ectopic insertion of the T-DNA (i.e. no deletion of RALF gene) and
the two ΔRALF transformants. The disease symptoms were recorded and disease scores
assigned as described by Rep et al. (2004), with scores of 0=healthy plant through to 4=
severely wilted plant.
No consistent significant differences in fungal virulence were found between wild
type Fol race 3 and any of the transformants (Fig. S7D-E, see Supporting Information).
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ΔRALF transformants not the ectopic mutant strain tested. Therefore, using a wound
infection assay, we could find no strong evidence that the RALF-B gene is involved in the
pathogenicity of Fol race 3 on tomato.
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DISCUSSION
Here we report that a number of fungal plant pathogens encode RALF-like peptides in
their genomes, and provide evidence that at least a subset of these peptides can be
perceived by plant cells. RALF peptides have high levels of conservation across a wide
range of plant species, and the importance of their roles in regulating plant physiology is
becoming increasingly apparent (Covey et al., 2010, Murphy & De Smet, 2014, Pearce et
al., 2001). The suggestion that fungi deploy RALF peptides as effectors with the assumed
consequence of improving fungal interaction with their hosts provides new perspective
both to understand the pathogenic strategies of fungi, and evolutionary mechanisms
underlying the acquisition of virulence.
Of the fungal genera carrying RALF homologues, the genus Fusarium has the most
diverse array with four groups of RALF homologues (Fig. 3), with two subtypes in
Groups I and IV. Each of the four groups has representatives in Fox, making Fox one of
the most interesting fungal species for further study of fungal RALF-peptides. However,
it is possible that this apparent diversity is in part due to the large number of Fusarium
species, and Fox formae speciales in particular, whose genomes are publicly available. In
bacteria, large numbers of genome sequences for a single species has catalysed the
concept of the pan-genome. The pan-genome represents the entire genomic coding
repertoire of all strains within a species enabling intra-specific variation to be observed
and assessed (Vernikos et al., 2015). This variation can be used to assist in explanations
of varied lifestyles between isolates. The idea of the pan-genome is becoming more
common in fungi and re-sequencing of fungal-genomes has begun to reveal the level of
intra-specific variation (Thynne et al., 2015a). With many re-sequenced Fox genomes
and diversity in Fox lifestyles (non pathogenic, plant pathogenic, insect pathogenic and
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opportunistic human pathogen), the genetic determinants underpinning this diversity
can be identified and analysed. Focusing on the RALF sequences identified here,
considerable intra-specific variation was observed between different Fox genomes.
Intriguingly, the two sequenced Fox isolates that are not are not reported plant
pathogens (isolate FOSC 3-a, which is a human clinical isolate, and isolate Fo47 which is
non-pathogenic) carry mutations in conserved cysteine residues that should render the
RALF peptide biologically inert. Disulphide bonds formed between the two pairs of
cysteines in RALF are essential for activity (Pearce et al., 2010). The RALF genes from
these isolates appear to be undergoing pseudogenization, perhaps because they are not
required outside of the plant pathogenic niche. In this study, the Fol RALF-B variant
elicited a strong response in the solanaceous plants tested. In contrast, there was a
comparatively negligible response to Fol RALF-C, with only weak activation of MAPK
observed in treated N. benthamiana. Perhaps these differences are related to the swap of
a conserved tyrosine to histidine early in the active peptide domain, as this tyrosine is
important for RALF activity (Pearce et al., 2010). RALF-C is found in Forl, which causes
crown rot of tomato, and therefore has a different pathogenic lifestyle to Fol. It is
possible that the RALF-C variant plays a different role in Forl than RALF-B does in Fol,
but why then does Fol MN25 carry RALF-C instead of RALF-B like Fol 4287? It is possible
that Fol MN25 carries other genetic factors, such as the Group IV RALF-like genes
present in Fol MN25 but absent from Fol 4287 (Fig. 3), that compensate for the absence
of RALF-B. These are questions for further study.
Although the RALF variation found within Fox does not reveal the origins of the RALF
gene family in fungi, their polyphyletic nature does suggest genetic mobility within Fox.
All four pairwise combinations of the two apparently mutually-exclusive subtypes of
Group I and Group IV RALF homologues (Fig. 3) were found in different formae speciales
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of Fox, suggesting that one or both of these genes may be mobile i.e. transmitted
horizontally. This opens up yet another area for further investigation.
Using a synthetic RALF-B peptide, we demonstrated that a fungal RALF peptide can
be perceived by plants. This is a highly significant result because it suggests that
pathogenic fungi hijack endogenous plant physiological mechanisms to enhance their
virulence. RALF was initially defined as a plant peptide able to trigger the alkalinisation
of plant cell cultures, and subsequently demonstrated to play varied roles in plant
development. (Pearce et al., 2001, Murphy & De Smet, 2014). Here we showed that
treatment of plants with both plant and fungal RALF caused seedling growth inhibition,
a rapid burst of ROS, MAPK activation and alkalinisation, all hallmarks of defence
activation by plants. Elicitation of ROS by RALFs has not been reported previously and
represents an important addition to the spectrum of responses associated with RALF
perception. We tested two representative divergent RALF sequences from the 12 F.
oxysporum sequences. Only one of these caused consistent activation of plant responses
at very low concentrations. Interestingly, RALF-B was very much more potent than the
endogenous Sl-RALF peptide in treated N. benthamiana, and showed marked differences
both in the extent and the timing of the plant response. It is interesting to note that Sl-
RALF only induced a strong response in tomato whereas RALF-B was able to induce a
strong response in both tomato and N. benthamiana. This is consistent with the
apparent conservation of RALF peptides across Fox spp.. In these species, these peptides
are located on the conserved (not lineage specific) chromosomes. As such, promiscuity
of activity among different potential hosts may be beneficial. At this stage it is difficult to
interpret the differences in activities between peptides, other than to point out that they
must be due to the sequence differences between the peptides. A key objective of this
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analysis was to demonstrate that a plant could perceive a fungal RALF peptide. This was
achieved.
The expression of RALF-B in Fol during in planta growth was consistent with a
potential role in mediating disease. However, under the conditions used for
pathogenicity assays in this study, strains of Fol lacking RALF-B displayed similar
disease symptoms to wild-type Fol implying that the peptide is not involved in the
induction of disease symptoms. These data suggest that RALF-B plays other as yet
undetermined roles in this host-pathogen interaction, not trialled in this study.
Inoculation was performed with cut roots, dipped in spore solution. This is a routine
technique for assaying F. oxysporum infections of tomato. Perhaps the fungal RALFs are
involved in manipulating the host prior to, and/or during colonisation. Potential roles
for RALF are the focus of on-going research.
Whilst this paper was still under review, Masachis et al. (2016) reported a functional
role for RALF-B in Fol pathogenicity and clear differences in the infection assays and
scoring criteria are apparent when compared to this study. Masachis et al. (2016) grew
plants for inoculation in vermiculite with no plant nutrients provided and it is possible
that assays conducted on such plants may have revealed an effect that was not evident
in healthy plants grown in potting mix well supplied with nutrients. They also scored
plant survival as opposed to disease symptoms and over a much longer period (up to 35
days) than we did (21 days) and it is interesting to note that their study also showed no
significant effects at 21 dpi i.e. no significant differences in survival. We are confident
therefore that we can conclude that, under the inoculation conditions and scoring
criteria used in this study, we could find no significant effect of the RALF-B knockout on
pathogenicity and can only conclude that any effect RALF-B had on pathogenicity was
too subtle for us to detect under our experimental conditions.
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An intriguing observation from the distribution of RALFs among fungi was that the
two poplar (P. trichocarpa) pathogens, Sphaerulina musiva (Mycosphaerella populorum)
and Septoria populina,(Mycosphaerella populicola) encode RALF27-like homologues.
Recent comparisons between these two species’ genomes uncovered an adaptation
event that likely altered the fitness of S. musiva to become more pathogenic to poplars,
in relation to S. populina (Dhillon et al., 2015). Similarly, the gain or evolution of this
peptide likely assisted interaction of these fungi with poplars. Unlike most of the other
fungal RALF-homologues identified, the most similar homologues to the S. musiva and S.
populina RALF27-like peptides are those of the host that they infect. This implies that
the fungal peptides have the same role in plants as the endogenous peptides. This is also
a potentially interesting model for studying the function of RALF27. No function has yet
been assigned to this member of the RALF family, which is only briefly mentioned in the
literature (Marmiroli & Maestri, 2014, Lafleur et al., 2015, Olsen et al., 2002).
Characterising the role of fungal RALF27 homologues during infection may also help
elucidate the function of the host homologue.
Similar to S. musiva and S. populina, both P. fijensis and M. phaseolina share RALF
homologues similar to their respective hosts. P. fijiensis has a RALF that resolves with
banana (Musa acuminate malaccensis), and M. phaseolina a RALF with sequence
similarity to a range of bean species. The RALF peptide from M. phaseolina is particularly
interesting as it shares extremely high levels of sequence conservation for the peptide
domain with a number of bean species (some of its primary hosts). However, this
sequence clearly relates to other fungal RALF peptides, falling with a group of peptides
shared among a range of fungal species (for example other members of the family
Botryosphaeriaceae and the genus Fusarium). This would suggest that the peptide
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domain of this protein in M. phaseolina has likely evolved to be better attuned with that
of its hosts.
The distribution of RALFs in the fungal kingdom is sporadic, with members
represented from the Dothideomycete and Sordariomycete classes, as well as in the
evolutionarily more distant Pucciniomycetes. Strikingly, the represented species are all
economically significant phytopathogens. The strong conservation of the RALF peptide
domain, and the in planta transcription and host perception of one such variant,
represents important evidence that fungi have co-opted this endogenous host signalling
system to enhance their fitness on plants. The question remains, however, what is the
ancestral origin of these conserved genes? The data presented here are consistent with
conflicting evolutionary scenarios; horizontal gene transfer (HGT) or convergent
evolution. At this time we cannot comment with any certainty on which scenario is the
most likely.
Although the primary focus of this analysis has been to present the findings that a
number of members of the fungal kingdom possess RALF peptides, we also identified a
limited number of plant-pathogenic bacteria which possess genes encoding amino acid
sequences with an incorporated RALF motif domain. However, we did not explore any
potential functional role for these proteins due to their striking differences from fungal
and plant RALFs. The mature peptides of the RALFs discussed in fungi are homologous
to the endogenous plant peptides. In contrast, the amino acid sequences in bacteria with
a RALF-like domain appear to be an amalgamation of a C-terminal RALF domain and the
S1 subunit of the pertussis toxin. Pertussis toxin has been studied in detail, particularly
its role as a virulence factor in bacterial-animal pathosystems. However, the function
these two unrelated domains may perform in bacterial/plant interactions is currently
unknown.
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There remains much to learn about the function of RALF peptides and their role in
plant growth and development. Adding to this mystery is how particular plant-
pathogenic organisms, both fungal and bacterial, utilise variants of these peptides to
facilitate host-infection and/or disease. In this study, we have demonstrated that
genomes of specific fungal and bacterial phytopathogens encode RALF peptides, of
which at least some are functionally active. We have focussed on the interaction
between Fol’s expression of a particular RALF peptide and infection in tomato. However,
there are a number of different RALF homologues utilised in various microorganism-
plant pathosystems. We anticipate that this discovery will form the basis of
understanding of how different RALF peptides are utilised by microorganisms to co-opt
plant-machinery to facilitate in planta growth.
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EXPERIMENTAL PROCEDURES
Identification of RALF homologues in fungal sequence databases
Homologues of RALF were identified with BLAST searches against the fungal sequence
databases in NCBI (http://www.ncbi.nlm.nih.gov) and available via the Mycocosm
portal in JGI (http://genome.jgi-psf.org/programs/fungi/index.jsf). The queries used
were the Arabidopsis thaliana plant RALF1 - RALF34 protein sequences from NCBI
(Table S1). Because of variability in the N-terminal and C-terminal regions, the BLAST
search queries were refined where needed to include only the mature RALF peptide
region (Pearce et al., 2010). BLASTp searches were also used to find RALF homologues
using fungal proteins with RALF annotations (Altschul et al., 1990). No e-value cut-off
was used. Instead, individual BLAST hits were assessed for the presence of RALF-
associated domains (Murphy & De Smet, 2014), regardless of e-value. tBLASTn searches
were used to find mRNA sequences, and un-annotated or differently-annotated RALF
homologues on fungal genome contigs/chromosomes (Gardiner et al. 2012; Ohm et al.
2012; Condon et al. 2013; Nemri et al. 2014). Where annotations were not present or
were different, the online Softberry server (http://linux1.softberry.com/berry.phtml)
was used to predict mRNA and protein sequences using the FGNESH+ prediction
algorithm (Solovyev et al., 2006). Fungal RALF protein sequences were used as queries
against the plant protein database in NCBI to find the top plant BLAST-hits (Altschul et
al., 1990).
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Alignment and analysis of RALF protein sequences
A representative database of plant RALF proteins was compiled by searching for any
plant amino acid sequence in the RefSeq database (Pruitt et al., 2007) labelled with the
term "RALF" or "Rapid Alkalinisation Factor". To compare RALF protein sequences from
fungi and plants, GLAM2 (Frith et al., 2008) was used to identify the conserved region
corresponding to the RALF domain from the plant sequences, conserved sites within the
RALF domain and to align the conserved sites. Its sister program GLAM2SCAN was then
used to align conserved sites from the fungal sequences, which were then added to the
plant alignment.
The Bayesian phylogenetics software ExaBayes (Aberer et al., 2014) was then used to
infer the posterior distribution of a RALF gene tree based on the aligned sequences. The
priors for this analysis were the LG substitution matrix (Le & Gascuel, 2008) (used as a
fixed amino acid model prior), and a uniform prior on tree topologies. Eight independent
chains were run with different seeds, sampled once every 3000 states. Using Tracer v1.6
(http://beast.bio.ed.ac.uk/Tracer), five chains were observed to converge at the same
likelihood after 1.8 million states, so sampled trees and statistics from the subsequent
3.6 million states of those converged chains were concatenated. The estimated sample
size of the concatenated log likelihood was 747, which suggests sufficient sampling of
the posterior, as that is much greater than the minimum threshold of 200 (Kuhner,
2009) .
A summary tree of 29 fungal RALF sequences and five plant RALF sequences was
generated by pruning each tree in the posterior distribution of RALF gene trees to just
those 34 tips, then producing an extended majority-rule tree from the posterior using
PAUP 4.0 (http://paup.csit.fsu.edu/). The resulting summary tree was rooted and
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ladderized using TreeGraph 2 (Stöver & Müller, 2010). The full summary tree of all
sequences was generated using the same method but without pruning. To produce a
combined phylogeny and alignment, the RALF sequences including non-conserved
residues were manually aligned, re-ordered to match the pruned summary tree and
individual amino acid residues coloured using JalView (Waterhouse et al., 2009).
Fusarium oxysporum RALF homologue sequences were aligned in Geneious 7.1.5
(Biomatters, New Zealand) using MUSCLE alignment (Edgar, 2004).
Synthetic peptides
Four synthetic RALF-like peptides were produced by Mimotopes (Australia) (Fig. S4, see
Supporting Information). First, synthetic tomato (Solanum lycopersici) RALF-like (Sl-
RALF) peptide (9.2mg with minimum purity of 96%) was chosen as a positive control
because of its similar domain structure to the RALF peptides described by Pearce et al
(2010) in a study describing important residues for activity and for its similarity to
Fusarium oxysporum f. sp. lycopersici (Fol) race 2. The synthetic peptide started at the
first amino acids reported to be required for RALF activity (“YISY”) (Pearce et al., 2010).
Second, inactive Sl-RALF peptide (Sl-RALF) was used as a negative control peptide
(9.4mg with minimum purity 82%). This peptide was designed missing the YISY motif
required for RALF function (Pearce et al., 2010). We synthesised two peptides of fungal
origin; Fol race 2 RALF (RALF-B) ( 9.3mg with minimum purity 86%) and Fol race 3
RALF (RALF-C) (9.2mg with minimum purity 77%). These two F. oxysporum RALF
peptide sequences are representative of the two main RALF variants observed in F.
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oxysporum (Fig. 3). All peptides were synthesized in oxidized form, with disulphide
bonds between cysteine residues at indicated positions (Fig. 2, Fig. 3).
Root inhibition in response to fungal-RALF synthetic peptides
Tomato seeds (Solanum lycopersicum cv. Moneymaker) were surface sterilized for 10
minutes in 4% sodium hypochlorite 10% ethanol and germinated in 2ml of 50% MS
media on an orbital shaker for two days. Three lots of three germinating seeds were
placed into new 2 mL 50% MS media with a final concentration of 10μM synthetic
peptide and incubated on the orbital shaker for 2 days. After this time, seedling weight
was measured and comparisons in developmental morphology made. Tukey's test was
used to determine which differences between treatments were significant for all
pairwise combinations of treatments, and to correct for multiple testing.
Measuring reactive oxygen species (ROS), the induction of alkalinisation and MAP
kinase production in tomato and Nicotiana benthamiana in response to synthetic
RALF peptides
ROS assays were performed as described previously (Segonzac et al., 2011), except that
L-012 (Wako chemicals) was used instead of luminol and luminescence was measured
on a TECAN plate reader, Infinite M200 PRO (Tecan). MAPK activation assays were
performed as described previously (Segonzac et al., 2011). For the alkalinisation
experiment, tomato or N. benthamiana leaves were cut the day before the assay in strips
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of approximately 1cm x 0.1cm and incubated in ddH2O overnight. The next day, 12-15
leaf strips of each were transferred to one well of a 12 well plate containing 2 mL of
sterile ddH2O. Leaves were incubated in the absence or presence 1 μM RALF peptide for
1 hr shaking at 120 rpm. The pH was measured using a pH electrode until the reading
was stable. The graphs show the average of three biological replicates and its estimated
standard error. Different letters depict significant differences using Tukey-Kramer HSD
test with a p-value smaller 0.01.
Reverse transcriptase PCR analysis of RALF-B expression during infection
Two-week-old susceptible tomato cv. M82 seedlings were inoculated by dipping their
roots in a suspension of 1x107 conidia per mL of Fol isolate #1943 or mock inoculated
by dipping in water. Fol-inoculated and mock-inoculated plants were then grown in a
controlled-environment growth room on a 25°C 16 hour day/20°C 8 hour night cycle
until collection of samples. Roots of 3-4 Fol-infected or mock-inoculated plants were
collected at 3 and 6 days post inoculation (dpi), washed with sterile deionised water,
pooled in a microcentrifuge tube and frozen in liquid nitrogen ready for RNA extraction.
Frozen root samples were ground in liquid nitrogen and total RNA was extracted using a
Plant RNeasy kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions.
Total RNA (2 μg) was treated with 2 μL of RQ1 RNase-Free DNase (Promega, Madison,
Wisconsin, USA) in a reaction volume of 20 μL containing 1x RQ1 DNAse reaction buffer
(400 mM Tris-HCl pH 8.0, 100 mM MgSO4, 10 mM CaCl2; Promega), and 1 μL of RNasin
ribonuclease inhibitor (Promega). The reaction was incubated at 37°C for 30 minutes,
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followed by an inactivation step at 65°C for 20 minutes. Treated RNA was reverse
transcribed into cDNA using Superscript III Reverse Transcriptase (Invitrogen, Carlsbad,
California, USA) and an oligo [dT]12-18 primer (Invitrogen) following the manufacturer’s
instructions. PCR (35 cycles) was carried out using Phire Hot Start II DNA Polymerase
(Thermo Fisher Scientific, Waltham, Massachusetts, USA) in a reaction volume of 25 μL
containing 1 μL of cDNA template as per the manufacturer’s instructions. Primers 5’
GCTGAAGCCAACCCCTATAA 3’ and 5’ TTACGATCCGGTTACCAAGC 3’ were used to
amplify RALFFol and primers 5’ AGCCTTACACCATCCGCTAC 3’ and 5’
CGCTGTAGTTGACCTCACCA 3’ were used to amplify FEM1 (Fusarium extracellular
matrix protein 1) (Schoffelmeer et al., 2001) as a positive control for fungal gene
expression. RNA and genomic DNA (50 ng) from non-inoculated tomato plants and five-
day-old cultures of Fol isolates #1943 and 4287 were used as controls. Controls also
included reactions in which reverse transcriptase or template DNA were omitted.
Fungal strains and pathogenicity test on tomato
The tomato M82 cultivar susceptible to Fol race 3 was used for pathogenicity tests.
Australian Fol race 3 isolate #1943 was used to generate RALF knockout mutants.
Pathogenicity tests were performed by the root-dip method (Wellman, 1939; Mes et al.,
1999). Using this method, roots of three-week-old plants of the tomato cultivar M82
(susceptible to Fol race 3) were wounded and subsequently inoculated with Fol race 3.
Disease symptoms were scored based on plant fresh weight and disease index as
described by Rep et al. (2004).
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Construction of Fol RALF deletion vectors
Flanking regions of the RALF gene were amplified from Fol race 3 genomic DNA using
primers RALF_FR1 and RALF_FR2 for the upstream flanking region and RALF_FR3 and
RALF_FR4 for the downstream flanking region (Table S2). The upstream flanking region
was cloned via KpnI and BsrGI restriction sites into the 5’ end of the hygromycin-
resistance cassette in the binary vector pPK2HPH (Michielse et al., 2009) and the
downstream flanking region was cloned via XbaI and HindIII sites into the 3’ end of the
hygromycin-resistance cassette, to generate pPK2HPH:ΔRALF.
A modified HSVtk gene cassette was generated and used as a second selection marker
as described by Khang et al. (2005). Briefly, the promoter of the trpC gene of A. nidulans
was amplified from the pGpdGFP binary vector (Sexton and Howlett, 2001) using the
primers trpC_p-F and trpC_p-R (Table S2) and cloned into the pGEMt easy vector
(Promega). The HSVtk gene was obtained from DNA of the herpes simplex virus (HSV)
isolate UL23 (provided by Prof. David Tscharke, The Australian National University)
using the primers HSVtk-F and HSVtk-R (Table S2) and cloned into the pGEMt:trpC
promoter intermediate vector via SpeI and SalI sites. The terminator sequence of Fol β-
tubulin gene was obtained from Fol genomic DNA using the primers βtub-F and βtub-R
and cloned into the pGEMt:trpC promoter:HSVtk coding sequence intermediate vector
via SalI and PmeI restriction sites. The entire HSVtk cassette was then transferred to the
pPK2HPH: ΔRALF vector and placed next to the RALF downstream flanking region,
using HindIII and PmeI sites (Fig. S6A, see Supporting Information).
Fol transformation was performed using the Agrobacterium tumefaciens strain
LBA4404 containing the appropriate binary vector. The transformation protocol was
adapted from Mullins et al. (2001) and Takken et al. (2004). The selection media for the
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RALF knockout mutants was supplemented with 75 μg/ml augmentin, 50 μg/ml
hygromycin and 5mM 5-fluoro-2’-deoxyuridine (F2dU). Deletion of the RALF gene was
confirmed by PCR using the primers scRALF_5’-F, RALF_int-R, RALF_int-F, scRALF_3’-R
and scGDP-R (Table S2 and Fig. S6B, see Supporting Information).
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ACKNOWLEDGEMENTS
The authors would like to thank Dr. Markus Albert for technical advice on the
alkalinisation assay. PSS is an Australian Research Council Future Fellow
(FT110100698). BS is supported by an Australian Research Council Discovery Early
Career Award (DE150101897). ET is supported by an Australian Postgraduate Award
and a Grains Research and Development Corporation Scholarship. ExaBayes was run on
a computer cluster managed by the Genome Discovery Unit of the Australian Cancer
Research Foundation’s Biomolecular Resource Facility.
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REFERENCES
Aberer, A., Kobert, K. and Stamatakis, A. (2014) ExaBayes: Massively parallel
bayesian tree inference for the whole-genome era. Mol. Biol. Evol. 31, 2553-2556.
Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic local
alignment search tool. J. Mol. Biol, 215, 403-410.
Bobay, B.G., DiGennaro, P., Scholl, E., Imin, N., Djordjevic, M.A. and Bird, D.M.
(2013) Solution NMR studies of the plant peptide hormone CEP inform function.
FEBS Letters, 587, 3979-3985.
Bömke, C. and Tudzynski, B. (2009) Diversity, regulation, and evolution of the
gibberellin biosynthetic pathway in fungi compared to plants and bacteria.
Phytochemistry, 70, 1876-1893.
Bournival, B., Scott, J. and Vallejos, C. (1989) An isozyme marker for resistance to race
3 of Fusarium oxysporum f. sp. lycopersici in tomato. Theor. Appl. Genet. 78, 489-
494.
Condon, B.J., Leng, Y., Wu, D., Bushley, K.E., Ohm, R.A., Otillar, R., Martin, J.,
Schackwitz, W., Grimwood, J., MohdZainudin, N., Xue, C., Wang, R., Manning,
V.A., Dhillon, B., Tu, Z.J., Steffenson, B.J., Salamov, A., Sun, H., Lowry, S.,
LaButti, K., Han, J., Copeland, A., Lindquist, E., Barry, K., Schmutz, J., Baker,
S.E., Ciuffetti, L.M., Grigoriev, I.V., Zhong, S. and Turgeon, B.G. (2013)
Comparative genome structure, secondary metabolite, and effector coding
capacity across Cochliobolus pathogens. PLoS Genet. e1003233
Cosgrove, D. J. (2000) Loosening of plant cell walls by expansins. Nature, 407, 321-326.
Covey, P.A., Subbaiah, C.C., Parsons, R.L., Pearce, G., Lay, F.T., Anderson, M.A., Ryan,
C.A. and Bedinger, P.A. (2010) A pollen-specific RALF from tomato that regulates
pollen tube elongation. Plant Physiol. 153, 703-715.
This article is protected by copyright. All rights reserved.
de Jonge, R., Bolton, M.D. and Thomma, B.P.H.J. (2011) How filamentous pathogens
co-opt plants: the ins and outs of fungal effectors. Curr. Opin. Plant Biol. 14, 400-
406.
de Jonge, R., van Esse, H.P., Maruthachalam, K., Bolton, M.D., Santhanam, P., Saber,
M. K., Zhang, Z., Usami, T., Lievens, B., Subbarao, K.V. and Thomma, B.P.
(2012) Tomato immune receptor Ve1 recognizes effector of multiple fungal
pathogens uncovered by genome and RNA sequencing. Proc. Nat. Acad. Sci. USA,
109, 5110-5115.
De Wit, P.J.G.M., Mehrabi, R., Van Den Burg, H.A. and Stergiopoulos, I. (2009) Fungal
effector proteins: Past, present and future: Review. Mol. Plant Path. 10, 735-747.
Delay, C., Imin, N. and Djordjevic, M.A. (2013) CEP genes regulate root and shoot
development in response to environmental cues and are specific to seed plants. J.
Exp. Bot. 64, 5383-5394.
Dhillon, B., Feau, N., Aerts, A.L., Beauseigle, S., Bernier, L., Copeland, A., Foster, A.,
Gill, N., Henrissat, B., Herath, P., LaButti, K.M., Levasseur, A., Lindquist, E.A.,
Majoor, E., Ohm, R.A., Pangilinan, J.L., Pribowo, A., Saddler, J.N., Sakalidis,
M.L., de Vries, R.P., Grigoriev, I.V., Goodwin, S.B., Tanguay, P. and Hamelin,
R.C. (2015) Horizontal gene transfer and gene dosage drives adaptation to wood
colonization in a tree pathogen. Proc. Nat. Acad. Sci. USA, 112, 3451-3456
Edgar, R.C. (2004) MUSCLE: Multiple sequence alignment with high accuracy and high
throughput. Nuc. Acids. Res. 32, 1792-1797.
Friesen, T.L., Stukenbrock, E.H., Liu, Z., Meinhardt, S., Ling, H., Faris, J.D.,
Rasmussen, J.B., Solomon, P.S., McDonald, B.A. and Oliver, R.P.(2006)
Emergence of a new disease as a result of interspecific virulence gene transfer.
Nat. Genet, 38, 953-956.
This article is protected by copyright. All rights reserved.
Frith, M.C., Saunders, N.F.W., Kobe, B. and Bailey, T.L. (2008) Discovering sequence
motifs with arbitrary insertions and deletions. PLoS Comput. Biol. 4, e1000071.
Gardiner, D.M., McDonald, M.C., Covarelli, L., Solomon, P.S., Rusu, A.G., Marshall, M.,
Kazan, K., Chakraborty, S., McDonald, B.A. and Manners, J.M.(2012)
Comparative pathogenomics reveals horizontally acquired novel virulence genes
in fungi infecting cereal hosts. PLoS Pathog. 8, e1002952.
Haruta, M., Sabat, G., Stecker, K., Minkoff, B.B. and Sussman, M.R. (2014) A peptide
hormone and its receptor protein kinase regulate plant cell expansion. Science,
343, 408-411.
Khang, C.H., Park, S.Y., Lee, Y.H. and Kang, S. (2005) A dual selection based, targeted
gene replacement tool for Magnaporthe grisea and Fusarium oxysporum. Fungal
Genet. Biol. 42, 483-492.
Klosterman, S.J., Subbarao, K.V., Kang, S., Veronese, P., Gold, S.E., Thomma, B.P.H. J.,
Chen, Z., Henrissat, B., Lee, Y-H., Park, J., Garcia-Pedrajas, M.D., Barbara, D.J.,
Anchieta, A., de Jonge, R., Santhanam, P., Maruthachalam, K., Atallah, Z.,
Amyotte, S.G., Paz, Z., Inderbitzin, P., Hayes, R.J., Heiman, D.I., Young, S.,
Zeng, Q., Engels, R., Galagan, J., Cuomo, C.A., Dobinson, K.F. and Ma, L.J.
(2011) Comparative genomics yields insights into niche adaptation of plant
vascular wilt pathogens. PLoS Pathog. 7, e1002137.
Kuhner, M.K. (2009) Coalescent genealogy samplers: windows into population history.
Trends Ecol. Evol. 24, 86-93.
Lafleur, E., Kapfer, C., Joly, V., Liu, Y., Tebbji, F., Daigle, C., Gray-Mitsumune, M.,
Cappadocia, M., Nantel, A., and Matton, D.P. (2016) The FRK1 mitogen-
activated protein kinase kinase kinase (MAPKKK) from Solanum chacoense is
This article is protected by copyright. All rights reserved.
involved in embryo sac and pollen development. J Exp Bot, (in press) doi:
10.1093/jxb/eru1524.
Le, S. and Gascuel, O. (2008) LG: An improved, general amino-acid replacement matrix.
Mol. Biol. Evol. 25, 1307-1320.
Ma, L.J., Van Der Does, H.C., Borkovich, K.A., Coleman, J.J., Daboussi, M.J., Di Pietro,
A., Dufresne, M., Freitag, M., Grabherr, M., Henrissat, B., Houterman, P.M.,
Kang, S., Shim, W.B., Woloshuk, C., Xie, X., Xu, J.R., Antoniw, J., Baker, S.E.,
Bluhm, B.H., Breakspear, A., Brown, D.W., Butchko, R.A.E., Chapman, S.,
Coulson, R., Coutinho, P.M., Danchin, E.G.J., Diener, A., Gale, L.R., Gardiner,
D.M., Goff, S., Hammond-Kosack, K.E., Hilburn, K., Hua-Van, A., Jonkers, W.,
Kazan, K., Kodira, C.D., Koehrsen, M., Kumar, L., Lee, Y.H., Li, L., Manners,
J.M., Miranda-Saavedra, D., Mukherjee, M., Park, G., Park, J., Park, S.Y.,
Proctor, R.H., Regev, A., Ruiz-Roldan, M.C., Sain, D., Sakthikumar, S., Sykes, S.,
Schwartz, D.C., Turgeon, B.G., Wapinski, I., Yoder, O., Young, S., Zeng, Q.,
Zhou, S., Galagan, J., Cuomo, C.A., Kistler, H.C. and Rep, M. (2010) Comparative
genomics reveals mobile pathogenicity chromosomes in Fusarium. Nature, 464,
367-373.
Marmiroli, N. and Maestri, E. (2014) Plant peptides in defense and signaling. Peptides,
56, 30-44.
Masachis, S., Segorbe, D., Turrà, D., Leon-Ruiz, M., Fürst, U., El Ghalid, M., Leonard,
G., López-Berges, M.S., Richards, T.A., Felix, G. and Di Pietro, A. (2016) A
fungal pathogen secretes plant alkalinizing peptides to increase infection. Nature
Microbiol. 1, 16043.
Mes, J.J., Weststeijn, E.A., Herlaar, F., Lambalk, J.J.M., Wijbrandi, J., Haring, M.A., and
Cornelissen, B.J.C. (1999) Biological and molecular characterization of Fusarium
This article is protected by copyright. All rights reserved.
oxysporum f. sp. lycopersici divides race 1 isolates into separate virulence groups.
Phytopathol. 89, 156-160.
Michielse, C.B., van Wijk, R., Reijnen, L., Cornelissen, B.J.C. and Rep, M. (2009)
Insight into the molecular requirements for pathogenicity of Fusarium oxysporum
f. sp. lycopersici through large-scale insertional mutagenesis. Genome Biol. 10, R4.
Mullins, E.D., Chen, X., Romaine, P., Raina, R., Geiser, D.M. and Kang, S. (2001)
Agrobacterium tumefaciens-mediated transformation of Fusarium oxysporum: an
efficient tool for insertional mutagenesis and gene transfer. Phytopathol. 91,173-
180.
Murphy, E. and De Smet, I. (2014) Understanding the RALF family: a tale of many
species. Trends Plant Sci. 19, 664-671.
Nemri ,A,. Saunders, D.G., Anderson, C., Upadhyaya, N.M., Win, J., Lawrence, G.J.,
Jones, D.A., Kamoun, S., Ellis, J.G. and Dodds, P.N. (2014) The genome
sequence and effector complement of the flax rust pathogen Melampsora lini.
Front. Plant Sci. 5, 98.
Nikolaidis, N., Doran, N. and Cosgrove, D.J. (2014) Plant expansins in bacteria and
fungi: evolution by horizontal gene transfer and independent domain fusion. Mol.
Biol. Evol. 31, 376-386.
Ohm, R.A., Feau, N., Henrissat, B., Schoch, C.L., Horwitz, B.A., Barry, K.W., Condon,
B.J., Copeland, A.C., Dhillon, B., Glaser, F., Hesse, C.N., Kosti, I., LaButti, K.,
Lindquist, E.A., Lucas, S., Salamov, A.A., Bradshaw, R.E., Ciuffetti, L., Hamelin,
R.C., Kema, G.H., Lawrence, C., Scott, J.A., Spatafora, J.W., Turgeon, B.G., de
Wit, P.J., Zhong, S., Goodwin, S.B. and Grigoriev, I.V. (2012) Diverse lifestyles
and strategies of plant pathogenesis encoded in the genomes of eighteen
Dothideomycetes fungi. PLoS Pathog. e1003037
This article is protected by copyright. All rights reserved.
Olsen, A.N., Mundy, J. and Skriver, K. (2002) Peptomics, identification of novel cationic
Arabidopsis peptides with conserved sequence motifs. In Silico Biology, 2, 441-
451.
Pearce, G., Moura, D.S., Stratmann, J. and Ryan, C.A. (2001) RALF, a 5-kDa ubiquitous
polypeptide in plants, arrests root growth and development. Proc. Nat. Acad. Sci.
USA, 98, 12843-12847.
Pearce, G., Yamaguchi, Y., Munske, G. and Ryan, C.A. (2010) Structure–activity studies
of RALF, Rapid Alkalinization Factor, reveal an essential–YISY–motif. Peptides,
31, 1973-1977.
Pruitt, K.D., Tatusova, T. and Maglott, D.R. (2007) NCBI reference sequences (RefSeq):
A curated non-redundant sequence database of genomes, transcripts and
proteins. Nuc. Acids Res. 35, D61-D65.
Rep, M., van der Does, H.C., Meijer, M., van Wijk, R., Houterman, P.M., Dekker, H.L.,
de Koster, C.G., and Cornelissen, B.J.C. (2004) A small, cysteine-rich protein
secreted by Fusarium oxysporum during colonization of xylem vessels is required
for I-3-mediated resistance in tomato. Mol. Microbiol. 53, 1373-1383.
Richards, T.A., Soanes, D.M., Jones, M.D., Vasieva, O., Leonard, G., Paszkiewicz, K.,
Foster, P.G., Hall, N. and Talbot, N.J. (2011) Horizontal gene transfer facilitated
the evolution of plant parasitic mechanisms in the oomycetes. Proc. Nat. Acad. Sci.
USA, 108, 15258-15263.
Schoffelmeer, E.A.M., Vossen, J.H., Van Doorn, A.A., Cornelissen, B.J.C. and Haring,
M.A. (2001) FEM1, a Fusarium oxysporum glycoprotein that is covalently linked
to the cell wall matrix and is conserved in filamentous fungi. Mol. Gen. Genet. 265,
143-152.
This article is protected by copyright. All rights reserved.
Segonzac, C., Feike, D., Gimenez-Ibanez, S., Hann, D.R., Zipfel, C. and Rathjen, J.P.
(2011) Hierarchy and roles of pathogen-associated molecular pattern-induced
responses in Nicotiana benthamiana. Plant Physiol. 156, 687-699.
Sexton, A.C. and Howlett, B.J. (2001) Green fluorescent protein as a reporter in the
Brassica-Leptosphaeria maculans interaction. Physiol. Mol. Plant Pathol. 58, 13-21.
Soanes, D. and Richards, T.A. (2014) Horizontal gene transfer in eukaryotic plant
pathogens. Ann. Rev. Phytopath. 52, 583-614.
Solovyev, V., Kosarev, P., Seledsov, I. and Vorobyev, D. (2006) Automatic annotation
of eukaryotic genes, pseudogenes and promoters. Genome Biol. 7, S10.11-12.
Srivastava, R., Liu, J., Guo, H., Yin, Y. and Howell, S. (2009) Regulation and processing
of a plant peptide hormone, AtRALF23, in Arabidopsis. Plant Journal, 59, 930-939.
Stöver, B.C. and Müller, K.F. (2010) TreeGraph 2: combining and visualizing evidence
from different phylogenetic analyses. BMC Bioinformatics, 11, 7.
Stukenbrock, E.H. and McDonald, B.A. (2008) The origins of plant pathogens in agro-
ecosystems. Ann. Rev. Phytopath. 46, 75-100.
Takken F.L.W., Van Wijk R., Michielse C.B., Houterman P.M., Ram A.F. and
Cornelissen B.J.C. (2004) A one-step method to convert vectors into binary
vectors suited for Agrobacterium-mediated transformation. Curr.Genet, 45, 242-
248.
Taylor, W. R. (1997) Residual colours: a proposal for aminochromography. Protein Eng.
10, 743-746.
Thynne, E., McDonald, M.C. and Solomon, P.S. (2015a) Phytopathogen emergence in
the genomics era. Trends Plant Sci. 20, 246-255.
This article is protected by copyright. All rights reserved.
Thynne, E., McDonald, M.C., Evans, M., Wallwork, H., Neate, S. and Solomon, P.S.
(2015b) Re-classification of the causal agent of white grain disorder on wheat as
three separate species of Eutiarosporella. Austral. Plant Path. 44, 527-539
Vernikos, G., Medini, D., Riley, D.R. and Tettelin, H. (2015) Ten years of pan-genome
analyses. Curr. Opin. Microbiol. 23, 148-154.
Waterhouse, A.M., Procter, J.B., Martin, D.M., Clamp, M. and Barton, G.J. (2009)
Jalview Version 2—a multiple sequence alignment editor and analysis
workbench. Bioinformatics, 25, 1189-1191.
Wellman, F.L. (1939) A technique for studying host resistance and pathogenicity in
tomato Fusarium wilt. Phytopathol. 29, 945-956.
Wolf, S. and Höfte, H. (2014) Growth control: A saga of cell walls, ROS, and peptide
receptors. Plant Cell, 26, 1848-1856.
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FIGURE LEGENDS
Fig. 1. Sequence of the Nicotiania tabacum RALF peptide (NtRALF). The underlined
region represents the predicted signal peptide whilst the arrow indicates the point of
cleavage by a subtilisin-like serine protease (Srivastava et al., 2009). The sequence in
red represents the active RALF peptide. Asterisks (*) denote the essential YSIY motif.
Cysteine residues required for disulphide bond formation and RALF peptide activity are
denoted by hashtags (#) (Pearce et al., 2010).
Fig. 2. Identification, alignment and analysis of the RALF domain in fungi and selected
plants. Aligned RALF peptide domain sequences from five isolates of Fusarium
oxysporum, 24 other fungal species and selected plants (highlighted in green) are
presented. Cysteine pairs expected to form disulphide bonds are labelled at the top. The
Melampsora lini RALF may have an alternative disulphide bond, marked with a dotted
line. Support values on each branch refer to the percentage posterior probability of each
clade. RALF sequences from two species of fungi, Septoria populicola and Sphaerulina
musiva, clustered with plant RALF27-like genes with 100% posterior support (labeled at
right). The plant RALF27-like sequences are from Populus trichocarpa (gi:566213100)
and Arabidopsis thaliana (gi:15230083). The other plant RALF sequences are from
Solanum lycopersicum (gi:460366641), corresponding to the peptide used here in
seedling and cell culture assays, from Nicotiana tomentosiformis (gi:697149758), which
is identical to the original RALF sequence reported from N. tabacum and A. thaliana
RALF1 (gi:15218637). The divergent RALF sequences of Fusarium oxysporum isolates
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4287 and MN25 are highlighted in gold. Individual amino acids are coloured using the
Taylor scheme (Taylor, 1997).
Fig. 3. Multiple sequence alignments of Fusarium RALF and RALF-like homologues.
Group I comprises a large family of RALF homologues from Fusarium verticillioides, F.
fujikuroi, F. circinata and various formae speciales of F. oxyporum (Fox). Note that the
Group IA RALF homologues FOWG_113369 Fox f. sp. lycopersici 4287 and FOCG_08085
Fox f. sp. radicis-lycopercici CL57 are very similar to one another, but divergent from the
Group IB RALF sequences at specific points within the predicted mature RALF peptide,
notably the sequence between the first two cysteines and a histidine substitution in
place of a conserved tyrosine (arrowed). Four cysteine residues involved in disulphide
bonding are conserved in all isolates except FOZG_15376, where the first cysteine is
swapped at position 39 to a tyrosine. FOYG_14526 has an early stop codon at site 67,
prior to the final cysteine at site 69. Group II comprise a small family of divergent RALF
homologues from Fox. f. sp. melonis and a human pathology isolate of Fox. Note that the f.
sp. melonis homologue has an apparent frameshift indicated by a slash (/) that could be
the consequence of a sequencing error rather than a frameshift mutation.
Group III comprises a small family of divergent RALF homologues from Fox f. sp. radicis-
lycopercici CL57, F. graminearum and F. pseudograminearum. Group IV comprises a large
family of RALF homologues that have an internal deletion relative to the RALF
homologues shown in groups I, II and III. This deletion includes the cysteine residues
that form the first disulphide bond indicated above the alignments. Note that this family
contains two apparently mutually exclusive sub-forms as can be seen from the
conserved residues highlighted in pink and blue.
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Conserved cysteines are highlighted in black. Other residues conserved between
Groups I and IV, and to some extent Groups II and III, are highlighted in green. Residues
conserved within Group I and to some extent Groups II and III are highlighted in yellow.
Residues conserved within Group IV and to some extent Groups II and III are highlighted
in red. Residues conserved within Group IV but differing between Groups IVA and IVB
are highlighted in pink and blue. Signal peptide cleavage sites were predicated using
SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/). Disulphide bond locations are
based on Pearce et al. (2001). ‡ indicates transcript and protein sequences predicted
using the same splice acceptor site as predicted for all of the other sequences in this
group. * denotes a stop codon.
Fig. 4. Effect of RALF peptides on germinating tomato seedlings.
A. Mean weights were calculated from whole seedlings grown in media containing 10
μM synthetic RALF peptides (n = 9). The letter codes a through d indicate statistical
significance: treatments which do not share any letter codes are significantly different (p
< 0.05, corrected for multiple testing). Error bars correspond to standard error.
B. Digital images of representative samples in A: a – Sl-RALF, b –Sl-RALFΔ, c – RALF-B; d
– RALF-C; e – Mock treatment.
Fig. 5. Synthetic RALF peptides induce ROS production and induce alkalinisation in N.
benthamiana and tomato leaves.
ROS burst over time after treatment with 10 μM of each synthetic RALF peptide in N.
benthamiana (A) and tomato (B) leaves. Panels C and D represent the induction of
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alkalinisation in N. benthamiana and tomato respectively by RALF-B and Sl-RALF. For A
and B, line colours are as follows: Dark blue – mock treatment; Red – Sl-RALF; Green –
RALF-B; Purple – RALF-C; Black –Sl-RALFΔ
Fig. 6. RT-PCR analysis shows that RALF-B is expressed during infection of tomato roots
by Fusarium oxysporum f. sp. lycopersici (Fol).
RT-PCR analysis of RALF-B (upper gel image) shows bands (expected size 123 bp)
consistent with gene expression in Fol-infected roots at 3 and 6 dpi but not in mock-
inoculated tomato plants or five-day-old mycelia grown in vitro. RT-PCR analysis of
FEM1 (lower gel image) shows bands (expected size 201 bp) consistent with expression
in infected roots at 3 and 6 dpi and five-day-old mycelia grown in vitro. The FEM1
control shows that cDNA synthesis was successful for the mycelial RNA sample and that
the resulting cDNA sample supported PCR. PCR analysis of a Fol genomic DNA sample
allowed detection of the RALF-B and FEM1 genes in Fol. The RALF-B and FEM1 genomic
DNA and cDNA products differ in size (expected sizes 179 bp versus 123 bp for RALF-B
and 250 bp versus 201 bp for FEM1), allowing the detection of genomic DNA
contamination in cDNA samples. No genomic RALF-B or FEM1 products were detected in
the cDNA samples.
Fig. S1. Gene tree of all identified RALF sequences from plants and fungi. This tree was
inferred using the RALF peptide domain amino acid sequences from 433 plant RALF
sequences and 29 fungal RALF sequences (highlighted in bold). Support values on each
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branch refer to the percentage posterior probability of each clade. Fungal RALF
sequences are labeled with the scientific name of the fungal species or isolate. Plant
RALF sequences are labeled with their NCBI GI code, binomial name and NCBI RefSeq
description. The tree was rooted to split the tree into RALF 27-like sequences and other
RALF sequences, a split with 100% posterior support.
Fig. S2. There are three divergent RALF groups within the Botryosphaeriaceae (A).
Particular motifs are unique among these groups (A). In particular, group BI has a motif
shared between few other species (plants and fungi) (B). One of M. phaseolina’s group
BII RALFs has a 100% identity for the mature peptide region with species of soy. These
are a primary host of M. phaseolina.
Fig. S3. A limited number of bacteria encode a protein with a pertussis toxin subunit
(S1) and a C-terminal RALF domain (A). This C-terminal RALF domain in these bacteria
shares homology with plant species RALFs (plant species outlined) (B).
Fig. S4. Amino acid sequences of peptides synthesized for the ROS burst and MAPK
activation assays
Fig. S5. Concentration dependent RALF-mediated production of ROS in N. benthamiana
leaves. (A) ROS burst over time upon treatment with 1 nM, 10 nM, 50 nM, 100 nM and
500 nM RALF-B. (B) ROS burst over time upon treatment with 100 nM, 1 µM and 10 µM
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of RALF-C Graphed data are ± SE, * P <0.05, **P <0.01, *** P < 0.001 (pairwise student’s
t-test comparing peptide treated to MOCK treated samples, n=6).
Fig S6. Activation of MAPKs in N. benthamiana leaves at 5 and 15 minutes after
treatment with the synthetic RALF peptides as indicated.
Fig. S7. Generation and screening screening of Fil race 3 transformants containing a
knockout of the RALF gene ( RALF). (A) Schematic representation of the RALF gene
replacement using the hygromycin phosphotransferase (hph) gene as a selectable
marker and the thymidine kinase (tk) gene as a counter-selectable marker. Arrows
indicate primers pairs used for the screening. (B) PCR detection of RALF gene using
scRALF 5’-F/RALF_int-R primers (primer set B; left gel image) and RALF_int-F/ scRALF
3’-R primers (primer set C; right gel image). Both gel images indicate absence of the
RALF coding sequence in the four RALF transformants. Fol = F. oxysporum f. sp.
lycopersic DNA. (C) PCR with scRALF 5’-F/sc_gdp-R primers (primer set D) indicates the
presence of the hph cassette in the RALF transformants. Ect = DNA from a
transformant with an ectopic T-DNA insertion in Fol. (D) Pathogenicity test on M82
plants with either Fol wild type race 3 (WT) or a Fol transformant with an ectopic T-
DNA insertion or Fol RALF transformants 2; 23, 24 and 31. Photographs
were taken 21 days post infection (dpi). (E) Distribution of disease scores for the
symptoms observed in two experiments for ten plants for each treatment at 21 dpi
(examples of plants contributing data for the upper graph are shown in D). Probability
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values were obtained using the one-tailed non-parametric Mann Whitney test to
determine significant differences (p ≤ 0.05) between treatments as indicated by
different letters.
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Table 1. Fungal species identified as containing RALF homologues
Fungi containing RALF sequences JGI/NCBI/GenBank/Ensembl/UniProt
sequence identifier
Ascomycetes
Dothideomycetes
Botryosphaeriaceae
Botryosphaeriaceae spp. See Table 3
Corynesporascaceae
Corynespora cassiicola jgi|Corca1|631921
Leptosphaeriaceae
Leptosphaeria biglobosa GenBank: FO905662.1
Leptosphaeria maculans NCBI Reference Sequence:
XP_003844940.1
Phoma tracheiphila jgi|Photr1|406253
Mycosphaerellaceae
Pseudocercospora fijiensis NCBI Reference Sequence:
XP_007921540.1
Septoria populicola jgi|Seppo1|99826
Sphaerulina musiva GenBank: EMF16709.1
Pleosporaceae
Pyrenophora tritici-repentis NCBI Reference Sequence:
XP_001937123.1
Pyrenophora teres jgi|Pyrtt1|194499:17017-24160
Setosphaeria turcica jgi|Settu1|scafd_12:1053528-1053731
Teratosphaeriaceae
Teratosphaeria nubilosa jgi|Ternu1|214030
Sordariomycetes
Glomerellaceae
Colletotrichum higginsianum GenBank: CCF44719.1
Plectosphaerellaceae
Verticillium dahliae GenBank: CP009075.1|
Verticillium alfalfae
Nectriaceae
Fusarium spp. See Table 2
Basidiomycetes
Pucciniomycetes
Melampsoraceae
Melampsora lini jgi|Melli1|sc_1450:29870-30703
Melampsora larici-populina NCBI Reference Sequence:
XP_007408986.1
Cronartiaceae
Cronartium quercuum jgi|Croqu1|50147
Page 49 of 57
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Table 2. Fusarium isolates and their RALF homologue IDs or DNA sequence origins.
Fusarium isolate Protein ID / origin
Fusarium fujikuroi IMI 58289
FFUJ_14827
chromosome 1:
6255677-6255856
Fusarium graminearum
-
1
(NRRL 31084)
FG05_30327
Fusarium oxysporum f. sp. conglutinans PHW808 (NRRL 54008) FOPG_11376
FOPG_06962
Fusarium oxysporum
f. sp.
conglutinans
Fo5176
FOXB_10100
FOXB_16123
Fusarium oxysporum
f. sp.
cubense
TR4 II5 (NRRL 54006)
FOIG_11494
FOIG_13631
Fusarium oxysporum
f. sp.
lycopersici
4287
(NRRL 34936)
FOXG_21151
Fusarium oxysporum
f. sp.
lycopersici
MN25 (NRRL 54003)
FOWG_11369
FOWG_10047
Fusarium oxysporum f. sp. melonis (NRRL 26406) FOMG_09497
FOMG_02269
Super Contig 597:
392-624
Fusarium oxysporum
f. sp.
pisi
HDV247 (NRRL 37622)
FOVG_12512
FOVG_16805
Fusarium oxysporum f. sp. radicis-lycopersici CL57 (NRRL 26381)
FOCG_08085
FOCG_16150
Super Contig 52:
30193-30411
Fusarium oxysporum
f. sp.
raphani
PHW815
(NRRL 54005)
FOQG_15667
FOQG_15928
Fusarium
oxysporum
f. sp.
vasinfectum
(NRRL 25433)
FOTG_09594
Fusarium oxysporum
human path. isolate FOSC 3
-
a (NRRL 32931)
FOYG_14526
FOYG_05840
Fusarium oxysporum
non
-
pathogenic Fo47 (NRRL 54002)
FOZG_15376
FOZG_02249
Fusarium pseudograminearum CS3096 Contig 007:
1344-1371
Fusarium verticillioides
7600 (NRRL 20956)
FVEG_11810
FVEG_14581
Fusarium circinata FSP34 Contig 00228:
8689-8910
Contig 00956:
4580-4747
Page 50 of 57
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Table 3. Botryosphaeriaceae isolates and their RALF homologue IDs or DNA
sequence origins.
Botryosphaeriaceae isolate Protein ID / origin
Botryosphaeria dothidea
Diplodia seriata
Macrophomina phaseolina
Neofusicoccum parvum
jgi|Botdo1_1|289944
Node_8012:
(92987-93247)
gi|821070376
gi|407928039
Contig_00502:
(91876-92412)
Contig_00151:
(16464-16099)
Contig_00254:
(124817-124602)
gi|615408450
Page 51 of 57
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Fig. 1. Sequence of the Nicotiania tabacum RALF peptide (NtRALF). The underlined region represents the
predicted signal peptide whilst the arrow indicates the point of cleavage by a subtilisin-like serine protease
(Srivastava et al., 2009). The sequence in red represents the active RALF peptide. Asterisks (*) denote the
essential YSIY motif. Cysteine residues required for disulphide bond formation and RALF peptide activity are
denoted by hashtags (#) (Pearce et al., 2010).
549x793mm (72 x 72 DPI)
Page 52 of 57
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Fig. 2. Identification, alignment and analysis of the RALF domain in fungi and selected plants. Aligned RALF
peptide domain sequences from five isolates of Fusarium oxysporum, 24 other fungal species and selected
plants (highlighted in gree
n) are presented. Cysteine pairs expected to form disulphide bonds are labelled at
the top. The Melampsora lini RALF may have an alternative disulphide bond, marked with a dotted line.
Support values on each branch refer to the percentage posterior probability of each clade. RALF sequences
from two species of fungi, Septoria populicola and Sphaerulina musiva, clustered with plant RALF27-like
genes with 100% posterior support (labeled at right). The plant RALF27-like sequences are from Populus
trichocarpa (gi:566213100) and Arabidopsis thaliana (gi:15230083). The other plant RALF sequences are
from Solanum lycopersicum (gi:460366641), corresponding to the peptide used here in seedling and cell
culture assays, from Nicotiana tomentosiformis (gi:697149758), which is identical to the original RALF
sequence reported from N. tabacum and A. thaliana RALF1 (gi:15218637). The divergent RALF sequences of
Fusarium oxysporum isolates 4287 and MN25 are highlighted in gold. Individual amino acids are coloured
using the Taylor scheme (Taylor, 1997).
Page 53 of 57
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549x793mm (72 x 72 DPI)
Page 54 of 57
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A
FOWG_11369 lycopersici MN25 MKFSILT-LSLITLAAA---APAAEPQSGVISHGALNRDHIPCSTEDGTKRNCRPGA----EANPYNQGCDAIAKCRGGVDGN*
FOCG_08085 radicis-lycopersici CL57 MKFSILT-LSLITLAAA---APAAEPQSGVISHGALNRDHIPCSTEDGTKRNCRPGA----EANPYNQGCDAIAKCRGGVDGN*
FOXG_21151 lycopersici 4287 MKFSIIT-LSLITLASA---APAAKPQSGEISYGALNRDHIPCSVKGASAANCRPGA----EANPYNRGCNAIEKCRGGVGGN*
FOMG_09497 melonis MKFSIIT-LSLITLASA---APAAKPQSGEISYGALNRDHIPCSVKGASAANCRPGA----EANPYNRGCNAIEKCRGGVGGN*
FOIG_11494 cubense II5 MKFSIIT-LSLITLASA---APAAKPQSGEISYGALNRDHIPCSVKGASAANCRPGA----EANPYNRGCNAIEKCRGGVGDN*
FOXB_10100 conglutinans Fo5176 MKFSIIT-LYLINLASA---APAAKPQSGEISYGALNRDHIPCSVKGASAANCRPGA----EANPYNRGCNAIEKCRGGVGDN*
FOPG_11376 conglutinans PHW808 MKFSIIT-LYLINLASA---APAAKPQSGEISYGALNRDHIPCSVKGASAANCRPGA----EANPYNRGCNAIEKCRGGVGDN*
FOVG_12512 pisi HDV247 MKFSIIT-LYLITLASA---APAAKPQSGEISYGALNRDHIPCSVKGASAANCRPGA----EANPYNRGCNAIEKCRGGVGDN*
FOTG_09594 vasinfectum MKFSIIT-LYLITLASA---APAAKPQSGEISYGALNRDHIPCSVKGASAANCRPGA----EANPYNRGCNAIEKCRGGVGDN*
FOQG_15667 raphani PHW815 MKFSIIT-LYLITLASA---APAAKPQSGEISYGALNRDHIPCSVKGASAANCRPGA----EANPYNRGCNAIEKCRGGVGDN*
FOZG_15376 non-pathogenic Fo47 MKFSIIT-LSLITLASA---APAAKPQSGEISYGALNRDHIPYSVKGASAANCRPGA----EANPYNRGCNAIEKCRGGVGDN*
FOYG_14526 human path. FOSC 3-a MKFSIIT-LSLITLASA---APAAKPQSGEISYGALNRDHIPCSVKGASAANCRPDA----EANPYNRGCNAI*KCCGGVGGN*
FVEG_11810 F. verticillioides MKFSIIT-LSLITLATA---APVAD-QGGEISYGALRHDNVPCSVRGASAANCHPGA----EANPYNRGCSAIEKCRGDVGNN*
FFUJ_14827 F. fujikuroi IMI 58289 MKFSIIT-LSLITLVTA---APAAAPQSGEISYGALKHDGVPCSLRGASAANCRPGA----EANPYNRGCSAIEKCRGGVGNN*
B
SC2: 5105820 human path. FOSC 3-a MKIFGPILVSLLAATVSA QNPNPGGPITFISYDALNKNRVPCSRRDGSIKNCYPAQGPYPPANSYTRGCSVIERCARPE*
SC597: 392 melonis MQVTGLFLIALGTISVSA QHSNPGV_ITFISYDAPRDNSIPCSRRGDYIKNCCPSGNVYPPPNTYTHGCTVAERCARPV*
C
C0007:1344 F. pseudograminearum MKFT-LAAFALISLAAA ---NPVAPRGGFISYDGLKRDGTPCSLKNESWQNCRPHAY----ANNWSRGCSPITRCRDG-
YPPGP*
FG05_30327 F. graminearum MKFS-IAALSLVTLAAA ---SPVQERTNYISYEGLKRDGTPCSLVTVSWQNCRPAAY----ANTWSRGCSPITRCRDG-
YPPGP*
SC52: 30193 radicis-lycopersici CL57 MKFSVITALTLVSFGAA ---------TQYISYEGLTRDGVPCDLRTVAWQNCRPKAY----
ANDWSRGCEAVFHCRGDDYPPGPPS*
D
FOWG_10047 lycopersici MN25 MKFFAVTILAMISGTLA---LPVAVPDNGHISYEGLKAP---------------PKA-PRQADDGYTRGCNPIFQCRGSV*
FOIG_13631 cubense II5 MKFFAVTILAMISGTLA---LPVAVPDNGHISYEGLKAP---------------PKA-PNQADDGYTRGCNPIFQCRGSV*
FOXB_16123 conglutinans Fo5176 MKFFAVTILAMISGTLA---LPVAVPDNGHISYEGLKAP---------------PKA-PNQADDGYTRGCNPIFQCRGSV*
FOPG_06962 conglutinans PHW808 MKFFAVTILAMISGTLA---LPVAVPDNGHISYEGLKAP---------------PKA-PNQADDGYTRGCNPIFQCRGSV*
FOVG_16805 pisi HDV247 MNFFAVTIFAMISGTLA---LPVAVPDNGHISYEGLKAP---------------PKA-PNQADDGYTRGCNPIFQCRGSV*
FOZG_02249 non-pathogenic Fo47 MKFFAVTILAMISGTLG---LPVAVPGNGHISYEGLKAP---------------PKA-PNQADDGYTRGCNPIFQCRGSV*
FOCG_16150 radicis-lycopersici CL57 MKFFTVTILAMVSGALA---MPVAAPNGGTINYEGLKGP-----------------SNTNPQPYKPSRPCLPSQQCRGKN*
FOMG_02269 melonis MKFFTVTILAMVSGALA---MPVAAPNGGTINYEGLKGP-----------------SNTNPQPYKPSRPCLPSQQCRGKN*
FOQG_15928 raphani PHW815 MKFFTVTILAMVSGALA---MPVAAPNGGTINYEGLKGP-----------------SNTNPQPYKPDRPCLPSQQCRGKN*
FOYG_05840 human path. FOSC 3-a MKFFTVTILAMVSGALA---MPVAAPNGGTINYEGLKGP-----------------SNTNPQPYKPDRPCLPSQQCRGKN*
FVEG_14581 F. verticillioides MKFFTLTILAMVSGALA---MPVAAPDGGTHNYNSLEGH-----------------GNPNPGPYKPDRPCLPSQQCRGKN*
chr1: 6255677 F. fujikuroi MKFFTLTILAMVSGALA---MPVAAPDNGYISYAGLEGG-----------------AKPHAGPYKPDRPCLPSQQCRGKN*
signal peptide
disulphide bond
disulphide bond
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Fig. 4. Effect of RALF peptides on germinating tomato seedlings.
A. Mean weights were calculated from whole seedlings grown in media containing 10 µM synthetic RALF
peptides (n = 9). The letter codes a through d indicate statistical significance: treatments which do not
share any letter codes are significantly different (p < 0.05, corrected for multiple testing). Error bars
correspond to standard error.
B. Digital images of representative samples in A: a – Sl-RALF, b –Sl-RALF∆, c – RALF-B; d – RALF-C; e –
Mock treatment.
549x793mm (72 x 72 DPI)
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Fig. 5. Synthetic RALF peptides induce ROS production and induce alkalinisation in N. benthamiana and
tomato leaves.
ROS burst over time after treatment with 10 µM of each synthetic RALF peptide in N. benthamiana (A) and
tomato (B) leaves. Panels C and D represent the induction of alkalinisation in N. benthamiana and tomato
respectively by RALF-B and Sl-RALF. For A and B, line colours are as follows: Dark blue – mock treatment;
Red – Sl-RALF; Green – RALF-B; Purple – RALF-C; Black –Sl-RALF∆
549x793mm (72 x 72 DPI)
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This article is protected by copyright. All rights reserved.
300 bp
100 bp
mock 3 dpi
mock 6 dpi
Fol
3 dpi
Fol
gDNA
Fol
mycelia
H
2
O
300 bp
100 bp
RALF
-
B
FEM1
Fol
6 dpi
cDNA
1 kb
1 kb
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... Molecular mimicry is a strategy employed by pathogens to colonize their hosts, in which pathogen molecules mechanistically mimic the functions of host components and modulate host responses to promote pathogenicity and fitness 89 . Homologues of plant RALFs were identified as widely distributed across phylogenetically distant fungi, including some economically important phytopathogens, such as Fusarium oxysporum and Fusarium graminearum [90][91][92] . The RALF-like genes are also present in multiple species of root-knot nematodes, including Meloidogyne incognita 93 . ...
... The RALF-like genes are also present in multiple species of root-knot nematodes, including Meloidogyne incognita 93 . Moreover, the RALF-like genes and genes carrying the RALF motif were predicted in some bacterial Escherichia coli strains and plant-pathogenic Actinobacteria 90,91 . The wide distribution of RALF-like sequences in different microorganisms across kingdoms suggests their importance in parasitism and association with the hosts. ...
... Phylogenetic and coalescent analyses indicate that RALF mimics from fungi and nematodes are intermixed with plant RALFs without an apparent evolutionary origin 90,91,93 . Most RALF-like sequences identified in pathogens are close to Arabidopsis RALF1. ...
Article
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Plant malectin-like receptor kinases (MLRs), also known as Catharanthus roseus receptor-like kinase-1-like proteins, are well known for their functions in pollen tube reception and tip growth, cell wall integrity sensing, and hormonal responses. Recently, mounting evidence has indicated a critical role for MLRs in plant immunity. Here we focus on the emerging functions of MLRs in modulating the two-tiered immune system mediated by cell-surface-resident pattern recognition receptors (PRRs) and intracellular nucleotide-binding leucine-rich repeat receptors (NLRs). MLRs complex with PRRs and NLRs and regulate immune receptor complex formation and stability. Rapid alkalinization factor peptide ligands, LORELEI-like glycosylphosphatidylinositol-anchored proteins and cell-wall-associated leucine-rich repeat extensins coordinate with MLRs to orchestrate PRR- and NLR-mediated immunity. We discuss the common theme and unique features of MLR complexes concatenating different branches of plant immune signalling.
... To date, among phytopathogenic fungi, the effector peptides which mimic peptide phytohormones of the RALF, PSK, IDA, and PEP families have been found: the RALF and PSK peptides are quite common in the fungal kingdom and were identified in more than 20 Basidiomycota and Ascomicota species, while the IDA and PEP peptides have been revealed in only a few species [41,72,73]. ...
... Furthermore, the RALF-like genes were identified in the genomes of 26 species of biotrophic and necrotrophic phytopathogenic fungi, which belong to the Pucciniomycetes class of the Basidiomycota and the Dothideomycetes and Sordariomycetes classes of the Ascomycota; some of these fungi species have even several RALF-like genes [72,73]. All of the fungi species which were found to possess the RALF genes are plant pathogens, indicating the role of RALFs in fungal pathogenesis [73]. ...
... Furthermore, the RALF-like genes were identified in the genomes of 26 species of biotrophic and necrotrophic phytopathogenic fungi, which belong to the Pucciniomycetes class of the Basidiomycota and the Dothideomycetes and Sordariomycetes classes of the Ascomycota; some of these fungi species have even several RALF-like genes [72,73]. All of the fungi species which were found to possess the RALF genes are plant pathogens, indicating the role of RALFs in fungal pathogenesis [73]. Most of the identified fungal RALF genes have sequence similarity to Arabidopsis AtRALF1 or AtRALF27. ...
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Citation: Dodueva, I.; Lebedeva, M.; Lutova, L. Dialog between Kingdoms: Enemies, Allies and Peptide Phytohormones. Plants 2021, 10, 2243. Abstract: Various plant hormones can integrate developmental and environmental responses, acting in a complex network, which allows plants to adjust their developmental processes to changing environments. In particular, plant peptide hormones regulate various aspects of plant growth and development as well as the response to environmental stress and the interaction of plants with their pathogens and symbionts. Various plant-interacting organisms, e.g., bacterial and fungal pathogens, plant-parasitic nematodes, as well as symbiotic and plant-beneficial bacteria and fungi, are able to manipulate phytohormonal level and/or signaling in the host plant in order to overcome plant immunity and to create the habitat and food source inside the plant body. The most striking example of such phytohormonal mimicry is the ability of certain plant pathogens and symbionts to produce peptide phytohormones of different classes. To date, in the genomes of plant-interacting bacteria, fungi, and nematodes, the genes encoding effectors which mimic seven classes of peptide phytohormones have been found. For some of these effectors, the interaction with plant receptors for peptide hormones and the effect on plant development and defense have been demonstrated. In this review, we focus on the currently described classes of peptide phytohormones found among the representatives of other kingdoms, as well as mechanisms of their action and possible evolutional origin.
... Since then, RALFs were found to be ubiquitous in plants and microbes. Genome-wide analyses have identified 37 RALFs in Arabidopsis (Abarca et al., 2021), 765 in 51 plant species (Campbell and Turner, 2017), 124 in 7 Rosaceae species , 17 in Fusarium species and 18 in 6 nematode species (Thynne et al., 2017;. The wide distribution of RALFs highlighted their functional importance. ...
... Rapid alkalinization factor was first associated with the plant defense response for its ability in MAPK activation (Pearce et al., 2001). Later, it was shown to be able to elicit ROS burst (Thynne et al., 2017), cytoplasmic Ca 2+ spikes and apoplastic pH ascending in Arabidopsis root cells (Gjetting et al., 2020), which are hallmarks of defense activation by plants. Here we found that the BnRALF10 peptide, derived from the C-terminus of BnPRORALF10, could stimulate ROS burst, intracellular Ca 2+ elevation, MAPK activation and defense-related gene expression induction (Figure 6). ...
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Rapid alkalinization factors (RALFs) were recently reported to be important players in plant immunity. Nevertheless, the signaling underlying RALF-triggered immunity in crop species against necrotrophic pathogens remains largely unknown. In this study, RALF family in the important oil crop oilseed rape (Brassica napus) was identified and functions of BnRALF10 in immunity against the devastating necrotrophic pathogen Sclerotinia sclerotiorum as well as the signaling underlying this immunity were revealed. The oilseed rape genome carried 61 RALFs, half of them were atypical, containing a less conserved YISY motif and lacking a RRXL motif or a pair of cysteines. Family-wide gene expression analyses demonstrated that patterns of expression in response to S. sclerotiorum infection and DAMP and PAMP treatments were generally RALF- and stimulus-specific. Most significantly responsive BnRALF genes were expressionally up-regulated by S. sclerotiorum, while in contrast, more BnRALF genes were down-regulated by BnPep5 and SsNLP1. These results indicate that members of BnRALF family are likely differentially involved in plant immunity. Functional analyses revealed that BnRALF10 provoked diverse immune responses in oilseed rape and stimulated resistance to S. sclerotiorum. These data support BnRALF10 to function as a DAMP to play a positive role in plant immunity. BnRALF10 interacted with BnFER. Silencing of BnFER decreased BnRALF10-induced reactive oxygen species (ROS) production and compromised rape resistance to S. sclerotiorum. These results back BnFER to be a receptor of BnRALF10. Furthermore, quantitative proteomic analysis identified dozens of BnRALF10-elicited defense (RED) proteins, which respond to BnRALF10 in protein abundance and play a role in defense. Our results revealed that BnRALF10 modulated the abundance of RED proteins to fine tune plant immunity. Collectively, our results provided some insights into the functions of oilseed rape RALFs and the signaling underlying BnRALF-triggered immunity.
... Endogenous Ralf-Feronia signaling in plants leads to inactivation of the plasma membrane H + -ATPase AHA2, which inhibits plant cell elongation [108]. The root-infecting fungus F. oxysporum is then able to induce alkalinization and cause plant disease using a functional homologue of the plant regulatory peptide Ralf, a peptide hormone capable of increasing the pH of surrounding fruit tissue or rhizosphere by more than two units, respectively, enhancing fungal colonization by increasing the pH of the apoplastic environment [109,110]. F. oxysporum mutants lacking a functional Ralf peptide fail to induce host alkalinization and exhibit markedly reduced virulence in tomato plants while eliciting a strong host immune response. F-Ralf appears to target the plant receptor-like kinase Feronia, which also mediates the response to endogenous plant Ralf peptides [111]. ...
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Plant pathogens are one of the main factors hindering the breeding of cash crops. Pathogens, including oomycetes, fungus, and bacteria, secrete effectors as invasion weapons to successfully invade and propagate in host plants. Here, we review recent advances made in the field of plant-pathogen interaction models and the action mechanisms of phytopathogenic effectors. The review illustrates how effectors from different species use similar and distinct strategies to infect host plants. We classify the main action mechanisms of effectors in plant-pathogen interactions according to the infestation process: targeting physical barriers for disruption, creating conditions conducive to infestation, protecting or masking themselves, interfering with host cell physiological activity, and manipulating plant downstream immune responses. The investigation of the functioning of plant pathogen effectors contributes to improved understanding of the molecular mechanisms of plant-pathogen interactions. This understanding has important theoretical value and is of practical significance in plant pathology and disease resistance genetics and breeding.
... Though RALFs are initially found and widely distributed in plants, recent studies have identified several RALF-likes in pathogens (Masachis et al., 2016;Thynne et al., 2017;Blackburn et al., 2020;Zhang et al., 2020a). The RALF-like from Fusarium oxysporum enhances fungi virulence, and Arabidopsis plants lacking FER are more resistant against F. oxysporum (Masachis et al., 2016). ...
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Soybean [ Glycine max (L.) Merr. ] is one of the most strategical oilseed crops that provides sustainable source of protein and oil worldwide. Cultivation of soybean is severely affected by root-knot nematode (RKN). However, the mechanism of RKN parasitism to soybeans is largely unknown. In this study, we identify GmLMM1 , which encodes a homolog of FERONIA-like receptor kinase in soybean, as a susceptible gene toward nematode. Mutations of GmLMM1 exhibit enhanced resistance against the RKN Meloidogyne incognita . RNA-sequencing (RNA-seq) analysis reveals a similar differential expression pattern for genes regulated by GmLMM1 ( Gmlmm1 vs. wild-type) and M. incognita ( M. incognita vs. mock), supporting the role of GmLMM1 in M. incognita infection. Unlike FERONIA in Arabidopsis , GmLMM1 specifically binds to Mi RALF1 and At RALF23 that suppress plant immunity, but not Mi RALF3 and At RALF1. Moreover, we found that the single-nucleotide polymorphism (SNP) in GmLMM1 leads to the natural resistance against RKNs in soybeans. Collectively, these findings uncover GmLMM1 as a susceptible target of nematode RALF-like 1 and provide new genetic resource for nematode resistant breeding.
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Canonical plant stress biology research has focused mainly on the dynamic regulation of internal genetic pathways in stress responses. Increasingly more studies suggest that plant‐mediated timely reshaping of the microbiota could also confer benefits in responding to certain biotic and abiotic stresses. This has led to the “cry for help” hypothesis, which is supported by the identification of plant genetic regulators integrating biotic/abiotic stress signaling and microbiota sculpting. Although diverse genetic mutants have been reported to affect microbiota composition, it has been challenging to confirm the causal link between specific microbiota changes and plant phenotypic outputs (e.g., fitness benefits) due to the complexity of microbial community composition. This limits the understanding of the relevance of plant‐mediated microbiota changes. We reviewed the genetic bases of host‐mediated reshaping of beneficial microbiota in response to biotic and abiotic stresses, and summarized the practical approaches linking microbiota changes and “functional outputs” in plants. Further understanding of the key regulators and pathways governing the assembly of stress‐alleviating microbiota would benefit the design of crops that could dynamically enlist beneficial microbiota under conditions of stress. Diverse plant genes are positively involved in reshaping the microbiota in response to stresses, which might be a novel layer of stress tolerance strategies during evolution. Plant‐associated microbiota affects diverse aspects of plant fitness and development, whereas how plants positively reshape a beneficial microbiota to enhance fitness is less studied. We reviewed the current understanding of the genetic mechanisms underlying plant‐mediated reshaping of the microbiota in response to stresses. Due to the complexity of the microbiota, it is challenging to confirm a causal link between microbiota changes and host phenotypes. We also reviewed the approaches to study the effect of microbiota changes on plant phenotypes. Plant‐associated microbiota affects diverse aspects of plant fitness and development, whereas how plants positively reshape a beneficial microbiota to enhance fitness is less studied. We reviewed the current understanding of the genetic mechanisms underlying plant‐mediated reshaping of the microbiota in response to stresses. Due to the complexity of the microbiota, it is challenging to confirm a causal link between microbiota changes and host phenotypes. We also reviewed the approaches to study the effect of microbiota changes on plant phenotypes.
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Small signalling peptides are key molecules for cell-to-cell communications in plants. The cysteine-rich signalling peptide, rapid alkalinisation factors (RALFs) family are involved in diverse developmental and stress responses and have expanded considerably during land plant evolution, implying neofunctionalisations in the RALF family. However, the ancestral roles of RALFs when land plant first acquired them remain unknown. Here, we functionally characterised two of the three RALFs in bryophyte Physcomitrium patens using loss-of-function mutants, overexpressors as well as fluorescent proteins tagged reporter lines. We showed that PpRALF1 and PpRALF2 have overlapping functions in promoting protonema tip growth and elongation, showing a homologous function as the Arabidopsis RALF1 in promoting root hair tip growth. Although both PpRALFs are secreted to the plasma membrane on which PpRALF1 symmetrically localised, PpRALF2 showed a polarized localisation at the growing tip. Notably, proteolytic cleavage of PpRALF1 is necessary for its function. Our data reveals a possible evolutionary origin of the RALF functions and suggests that functional divergence of RALFs is essential to drive complex morphogenesis and to facilitate other novel processes in land plants.
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