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

June 2016
Molecular Plant Pathology 18(6)

DOI:10.1111/mpp.12444

Authors:

Elisha Thynne

The Sainsbury Laboratory

Jaime Simbaqueba

SINCHI

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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.

Identification, alignment and analysis of the RALF domain in fungi and selected

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Multiple sequence alignments of Fusarium RALF and RALF-like homologues.

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Effect of RALF peptides on germinating tomato seedlings.

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Fig. S1. Gene tree of all identified RALF sequences from plants and fungi. This tree was

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Fig. S4. Amino acid sequences of peptides synthesized for the ROS burst and MAPK

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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

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.

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

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

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.

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,

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

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-

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).

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.

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).

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

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).

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.

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

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

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

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.

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

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.

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.

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).

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

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.

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

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,

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).

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

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).

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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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

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.

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

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

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

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

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

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.

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

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

(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

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

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

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

549x793mm (72 x 72 DPI)

Page 54 of 57

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*

SC2: 5105820 human path. FOSC 3-a MKIFGPILVSLLAATVSA QNPNPGGPITFISYDALNKNRVPCSRRDGSIKNCYPAQGPYPPANSYTRGCSVIERCARPE*

SC597: 392 melonis MQVTGLFLIALGTISVSA QHSNPGV_ITFISYDAPRDNSIPCSRRGDYIKNCCPSGNVYPPPNTYTHGCTVAERCARPV*

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*

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

Page 55 of 57

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)

Page 56 of 57

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)

Page 57 of 57

300 bp

100 bp

mock 3 dpi

mock 6 dpi

Fol

3 dpi

Fol

gDNA

Fol

mycelia

300 bp

100 bp

RALF

FEM1

Fol

6 dpi

cDNA

1 kb

Page 58 of 57

RALF peptides modulate immune response in the moss Physcomitrium patens

Article

Full-text available

Jan 2023

Background RAPID ALKALINIZATION FACTOR (RALFs) are cysteine-rich peptides that regulate multiple physiological processes in plants. This peptide family has considerably expanded during land plant evolution, but the role of ancient RALFs in modulating stress responses is unknown.Results: Here, we used the moss Physcomitrium patens as a model to gain insight into the role of RALF peptides in the coordination of plant growth and stress response in non-vascular plants. The quantitative proteomic analysis revealed concerted downregulation of M6 metalloprotease and some membrane proteins, including those involved in stress response, in PpRALF1, 2 and 3 knockout (KO) lines. The subsequent analysis revealed the role of PpRALF3 in growth regulation under abiotic and biotic stress conditions, implying the importance of RALFs in responding to various adverse conditions in bryophytes. We found that knockout of the PpRALF2 and PpRALF3 genes resulted in increased resistance to bacterial and fungal phytopathogens, Pectobacterium carotovorum and Fusarium solani, suggesting the role of these peptides in negative regulation of the immune response in P. patens. Comparing the transcriptomes of PpRALF3 KO and wild-type plants infected by F. solani showed that the regulation of genes in the phenylpropanoid pathway and those involved in cell wall modification and biogenesis was different in these two genotypes. Conclusion Thus, our study sheds light on the function of the previously uncharacterized PpRALF3 peptide and gives a clue to the ancestral functions of RALF peptides in plant stress response.

Management of Fusarium Wilt Disease of Maize (Zea mays L.) with Selected Antimycotic Plant Extracts and Inhibitory Bacillus Strains

Article

Full-text available

May 2023
CURR MICROBIOL

Maize (Zea mays L. var. Everta) is an economically significant crop cultivated worldwide. Unfortunately, microbial diseases, especially, mycopathogens such as Fusarium species, militate against the production of maize. Protective microbial species and bioactive plant extracts have been studied in the control of plant pathogens. However, there is a dearth of information on their comparative efficacy and effect on wilt disease of maize caused by Fusarium solani, as investigated in this study. Using the ITS and 16s rDNA primers for fungal pathogen and bacterial strains, respectively, the mycopathogen was confirmed as Fusarium solani FCI20, while biocontrol Bacillus strains were identified as Bacillus velezensis EBs02 and Bacillus thuringiensis EBs04. Fusarium solani FCI20 successfully infected maize seedlings through rhizosphere inoculation, and caused severe leaf chlorosis, necrosis and wilt in maize seedlings. Bacillus thuringiensis EBs04 expressed the highest in vitro mycelial inhibition (85.20%), followed by Gmelina arborea (78.58%), while Milicia excelsa appeared to express the lowest mycelial inhibition potential (49.95%). Bacillus velezensis EBs02 expressed the highest in vivo disease severity reduction in maize seedlings (with percentage disease control of 84.16), while B. thuringiensis-treated plants had the lowest incidence of wilt disease (4.32%). However, contrary to their in vitro mycelial inhibition potential, B. velezensis EBs02, Gmelina arborea, Milicia excelsa and Cola nitida expressed radically different levels of in vivo wilt disease control in maize seedlings. Consequent upon the biocontrol patterns observed in this study, in vivo assay should be considered in the preliminary selection of promising biocontrol agents against phytopathogens, such as Fusarium species.

The Phaseolus vulgaris Receptor-Like Kinase PvFER1 and the Small Peptides PvRALF1 and PvRALF6 Regulate Nodule Number as a Function of Nitrate Availability

Article

Full-text available

Mar 2023
INT J MOL SCI

Legumes associate with Gram-negative soil bacteria called rhizobia, resulting in the formation of a nitrogen-fixing organ, the nodule. Nodules are an important sink for photosynthates for legumes, so these plants have developed a systemic regulation mechanism that controls their optimal number of nodules, the so-called autoregulation of nodulation (AON) pathway, to balance energy costs with the benefits of nitrogen fixation. In addition, soil nitrate inhibits nodulation in a dose-dependent manner, through systemic and local mechanisms. The CLE family of peptides and their receptors are key to tightly controlling these inhibitory responses. In the present study, a functional analysis revealed that PvFER1, PvRALF1, and PvRALF6 act as positive regulators of the nodule number in growth medium containing 0 mM of nitrate but as negative regulators in medium with 2 and 5 mM of nitrate. Furthermore, the effect on nodule number was found to be consistent with changes in the expression levels of genes associated with the AON pathway and with the nitrate-mediated regulation of nodulation (NRN). Collectively, these data suggest that PvFER1, PvRALF1, and PvRALF6 regulate the optimal number of nodules as a function of nitrate availability.

Genome-wide characterization of soybean RALF genes and their expression responses to Fusarium oxysporum

Article

Full-text available

Oct 2022

RALFs (RAPID ALKALINIZATION FACTORs) are small peptides required for plant growth, development and immunity. RALF has recently been discovered to regulate plant resistance to fungal infection. However, little is known in crops, particularly in soybean. Here, 27 RALFs were identified in the genome of Glycine max . All Glycine max RALFs ( GmRALFs ) and 34 Arabidopsis RALFs were classified into 12 clades via the phylogenetic analyses. Gene structures, conserved motifs, chromosome distribution and cis -elements were analyzed in this study. Furthermore, 18 GmRALFs were found in response to Fusarium oxysporum ( F. oxysporum ) infection in soybean and to have distinct expression patterns. Among them, secretory function of two GmRALFs were identified, and three GmRALFs were detected to interact with FERONIA in Glycine max (GmFERONIA, GmFER). Our current study systematically identified and characterized GmRALFs in the soybean genome, laying a groundwork for further functional analyses and soybean breeding.

Genome-Wide Identification and Comparative Analysis of RALF Gene Family in Legume and Non-Legume Species

Article

Full-text available

May 2023
INT J MOL SCI

Rapid alkalinization factor (RALF) are small secreted peptide hormones that can induce rapid alkalinization in a medium. They act as signaling molecules in plants, playing a critical role in plant development and growth, especially in plant immunity. Although the function of RALF peptides has been comprehensively analyzed, the evolutionary mechanism of RALFs in symbiosis has not been studied. In this study, 41, 24, 17 and 12 RALFs were identified in Arabidopsis, soybean, Lotus and Medicago, respectively. A comparative analysis including the molecular characteristics and conserved motifs suggested that the RALF pre-peptides in soybean represented a higher value of isoelectric point and more conservative motifs/residues composition than other species. All 94 RALFs were divided into two clades according to the phylogenetic analysis. Chromosome distribution and synteny analysis suggested that the expansion of the RALF gene family in Arabidopsis mainly depended on tandem duplication, while segment duplication played a dominant role in legume species. The expression levels of most RALFs in soybean were significantly affected by the treatment of rhizobia. Seven GmRALFs are potentially involved in the release of rhizobia in the cortex cells. Overall, our research provides novel insights into the understanding of the role of the RALF gene family in nodule symbiosis.

Assembling Quality Genomes of Flax Fungal Pathogens from Oxford Nanopore Technologies Data

Article

Full-text available

Feb 2023
J. Fungi

Flax (Linum usitatissimum L.) is attacked by numerous devastating fungal pathogens, including Colletotrichum lini, Aureobasidium pullulans, and Fusarium verticillioides (Fusarium moniliforme). The effective control of flax diseases follows the paradigm of extensive molecular research on pathogenicity. However, such studies require quality genome sequences of the studied organisms. This article reports on the approaches to assembling a high-quality fungal genome from the Oxford Nanopore Technologies data. We sequenced the genomes of C. lini, A. pullulans, and F. verticillioides (F. moniliforme) and received different volumes of sequencing data: 1.7 Gb, 3.9 Gb, and 11.1 Gb, respectively. To obtain the optimal genome sequences, we studied the effect of input data quality and genome coverage on assembly statistics and tested the performance of different assembling and polishing software. For C. lini, the most contiguous and complete assembly was obtained by the Flye assembler and the Homopolish polisher. The genome coverage had more effect than data quality on assembly statistics, likely due to the relatively low amount of sequencing data obtained for C. lini. The final assembly was 53.4 Mb long and 96.4% complete (according to the glomerellales_odb10 BUSCO dataset), consisted of 42 contigs, and had an N50 of 4.4 Mb. For A. pullulans and F. verticillioides (F. moniliforme), the best assemblies were produced by Canu–Medaka and Canu–Homopolish, respectively. The final assembly of A. pullulans had a length of 29.5 Mb, 99.4% completeness (dothideomycetes_odb10), an N50 of 2.4 Mb and consisted of 32 contigs. F. verticillioides (F. moniliforme) assembly was 44.1 Mb long, 97.8% complete (hypocreales_odb10), consisted of 54 contigs, and had an N50 of 4.4 Mb. The obtained results can serve as a guideline for assembling a de novo genome of a fungus. In addition, our data can be used in genomic studies of fungal pathogens or plant–pathogen interactions and assist in the management of flax diseases.

Mechanisms of plant cell wall surveillance in response to pathogens, cell wall-derived ligands and the effect of expansins to infection resistance or susceptibility

Article

Full-text available

Aug 2022

Cell wall integrity is tightly regulated and maintained given that non-physiological modification of cell walls could render plants vulnerable to biotic and/or abiotic stresses. Expansins are plant cell wall-modifying proteins active during many developmental and physiological processes, but they can also be produced by bacteria and fungi during interaction with plant hosts. Cell wall alteration brought about by ectopic expression, overexpression, or exogenous addition of expansins from either eukaryote or prokaryote origin can in some instances provide resistance to pathogens, while in other cases plants become more susceptible to infection. In these circumstances altered cell wall mechanical properties might be directly responsible for pathogen resistance or susceptibility outcomes. Simultaneously, through membrane receptors for enzymatically released cell wall fragments or by sensing modified cell wall barrier properties, plants trigger intracellular signaling cascades inducing defense responses and reinforcement of the cell wall, contributing to various infection phenotypes, in which expansins might also be involved. Here, we review the plant immune response activated by cell wall surveillance mechanisms, cell wall fragments identified as responsible for immune responses, and expansin’s roles in resistance and susceptibility of plants to pathogen attack.

Rapid alkalinization factor: function, regulation, and potential applications in agriculture

Article

Full-text available

May 2023

Rapid alkalinization factor (RALF) is widespread throughout the plant kingdom and controls many aspects of plant life. Current studies on the regulatory mechanism underlying RALF function mainly focus on Arabidopsis, but little is known about the role of RALF in crop plants. Here, we systematically and comprehensively analyzed the relation between RALF family genes from five important crops and those in the model plant Arabidopsis thaliana . Simultaneously, we summarized the functions of RALFs in controlling growth and developmental behavior using conservative motifs as cues and predicted the regulatory role of RALFs in cereal crops. In conclusion, RALF has considerable application potential in improving crop yields and increasing economic benefits. Using gene editing technology or taking advantage of RALF as a hormone additive are effective way to amplify the role of RALF in crop plants.

Phyllosphere Microbiome

Article

Feb 2022
ANNU REV PLANT BIOL

The aboveground parts of terrestrial plants are colonized by a variety of microbes that collectively constitute the phyllosphere microbiota. Decades of pioneering work using individual phyllosphere microbes, including commensals and pathogens, have provided foundational knowledge about how individual microbes adapt to the phyllosphere environment and their role in providing biological control against pathogens. Recent studies have revealed a more complete repertoire of phyllosphere microbiota across plant taxa and how plants respond to and regulate the level and composition of phyllosphere microbiota. Importantly, the development of several gnotobiotic systems is allowing causative and mechanistic studies to determine the contributions of microbiota to phyllosphere health and productivity. New insights into how the phyllosphere carries out key biological processes, including photosynthesis, biomass accumulation, reproduction, and defense against biotic and abiotic insults, in either the presence or absence of a normal microbiota could unleash novel plant- and microbiota-based technologies to improve agriculturally relevant traits of crop plants. Expected final online publication date for the Annual Review of Plant Biology, Volume 74 is May 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

RALF peptides modulate immune response in the moss Physcomitrium patens

Preprint

Oct 2022

RAPID ALKALINIZATION FACTOR (RALFs) are cysteine-rich peptides that regulate multiple physiological processes in plants. This peptide family has considerably expanded during land plant evolution, but the role of ancient RALFs in modulating stress response is unknown. Here, we used the moss Physcomitrium patens as a model to gain insight into the role of RALF peptides in coordination of plant growth and stress response in non-vascular plants. The quantitative proteomic analysis revealed concerted downregulation of M6 metalloproteases and some membrane proteins, including those involved in stress response, in PpRALF1, 2 and 3 knockout (KO) lines. We found that knockout of PpRALF2 and PpRALF3 genes resulted in increased resistance to bacterial and fungal phytopathogens - Pectobacterium carotovorum and Fusarium solani , suggesting the role of these peptides in negative regulation of immune response in P. patens . The comparative transcriptome analysis of PpRALF3 KO and wild type plants under Fusarium solani infection showed the clear difference in regulation of genes belonging to phenylpropanoid pathway and associated with cell wall modification and biogenesis between these genotypes. The follow-up analysis revealed the role of PpRALF3 in growth regulation under abiotic and biotic stress regulation, which suggests the role of RALFs in responses to different adverse conditions. Thus, our study sheds light on the function of the previously uncharacterized PpRALF3 peptide and gives a clue to ancestral functions of RALF peptides in plant stress response.

Re-classification of the causal agent of white grain disorder on wheat as three separate species of Eutiarosporella

Article

Full-text available

Aug 2015
AUSTRALAS PLANT PATH

In the late 1990s, a novel Botryosphaeria-like fungal pathogen was observed causing a disease on wheat in Queensland, characterised as white grain disorder (WGD). In recent years, this disease has sporadically appeared across the eastern states of Australia. In this study, internal transcribed spacer (ITS) region sequences were used to compare these fungi to other Botryosphaeriaceae spp. to show that they should be reclassified as members of the Eutiarosporella genus. Using a small population of WGD isolates, we built a three-loci maximum likelihood tree, using ITS, β-tubulin, and Elongation Factor1-α sequences to show that there are three separate Eutiarosporella spp. found in infected grain. This multigene tree, with the support of phenotypic differences between clades observed in vitro, show that that the causal agents of WGD should be delimited into three divergent species; Eutiarosporella tritici-australis, Eutiarosporella darliae, and Eutiarosporella pseudodarliae.

Horizontal gene transfer and gene dosage drives adaptation to wood colonization in a tree pathogen

Article

Full-text available

Mar 2015

Significance Some of the most damaging tree diseases are caused by pathogens that induce cankers, a stem deformation often lethal. To investigate the cause of this adaptation, we sequenced the genomes of poplar pathogens that do and do not cause cankers. We found a unique cluster of genes that produce secondary metabolites and are co-activated when the canker pathogen is grown on poplar wood and leaves. The gene genealogy is discordant with the species phylogeny, showing a signature of horizontal transfer from fungi associated with wood decay. Furthermore, genes encoding hemicellulose-degrading enzymes are up-regulated on poplar wood chips, with some having been acquired horizontally. We propose that adaptation to colonize poplar woody stems is the result of acquisition of these genes.

The FRK1 mitogen-activated protein kinase kinase kinase (MAPKKK) from Solanum chacoense is involved in embryo sac and pollen development

Article

Full-text available

Jan 2015

The fertilization-related kinase 1 (ScFRK1), a nuclear-localized mitogen-activated protein kinase kinase kinase (MAPKKK) from the wild potato species Solanum chacoense, belongs to a small group of pMEKKs that do not possess an extended N- or C-terminal regulatory domain. Initially selected based on its highly specific expression profile following fertilization, in situ expression analyses revealed that the ScFRK1 gene is also expressed early on during female gametophyte development in the integument and megaspore mother cell and, later, in the synergid and egg cells of the embryo sac. ScFRK1 mRNAs are also detected in pollen mother cells. Transgenic plants with lower or barely detectable levels of ScFRK1 mRNAs lead to the production of small fruits with severely reduced seed set, resulting from a concomitant decline in the number of normal embryo sacs produced. Megagametogenesis and microgametogenesis were affected, as megaspores did not progress beyond the functional megaspore (FG1) stage and the microspore collapsed around the first pollen mitosis. As for other mutants that affect embryo sac development, pollen tube guidance was severely affected in the ScFRK1 transgenic lines. Gametophyte to sporophyte communication was also affected, as observed from a marked change in the transcriptomic profiles of the sporophytic tissues of the ovule. The ScFRK1 MAPKKK is thus involved in a signalling cascade that regulates both male and female gamete development.

Ten years of pan-genome analyses

Article

Full-text available

Dec 2014
CURR OPIN MICROBIOL

ExaBayes: Massively Parallel Bayesian Tree Inference for the Whole-Genome Era

Article

Full-text available

Aug 2014
MOL BIOL EVOL

Modern sequencing technology now allows biologists to collect the entirety of molecular evidence for reconstructing evolutionary trees. We introduce a novel, user-friendly software package engineered for conducting state-of-the-art Bayesian tree inferences on data sets of arbitrary size. Our software introduces a nonblocking parallelization of Metropolis-coupled chains, modifications for efficient analyses of data sets comprising thousands of partitions and memory saving techniques. We report on first experiences with Bayesian inferences at the whole-genome level using the SuperMUC supercomputer and simulated data.

Horizontal Gene Transfer in Eukaryotic Plant Pathogens

Article

Full-text available

Aug 2014

Gene transfer has been identified as a prevalent and pervasive phenomenon and an important source of genomic innovation in bacteria. The role of gene transfer in microbial eukaryotes seems to be of a reduced magnitude but in some cases can drive important evolutionary innovations, such as new functions that underpin the colonization of different niches. The aim of this review is to summarize published cases that support the hypothesis that horizontal gene transfer (HGT) has played a role in the evolution of phytopathogenic traits in fungi and oomycetes. Our survey of the literature identifies 46 proposed cases of transfer of genes that have a putative or experimentally demonstrable phytopathogenic function. When considering the life-cycle steps through which a pathogen must progress, the majority of the HGTs identified are associated with invading, degrading, and manipulating the host. Taken together, these data suggest HGT has played a role in shaping how fungi and oomycetes colonize plant hosts.

Understanding the RALF family: A tale of many species

Article

Full-text available

Jul 2014
TRENDS PLANT SCI

Small secreted peptides are gaining importance as signalling molecules in plants. Among the 1000 open reading frames (ORFs) in the Arabidopsis (Arabidopsis thaliana) genome potentially encoding small secreted peptides, the members of the RAPID ALKALINIZATION FACTOR (RALF) family of peptides have been linked to several physiological and developmental processes. Here, we provide a comprehensive overview of current knowledge on the RALF family. Discovered in tobacco (Nicotiana tabacum), the role of RALF peptides has been investigated in numerous plant species. Together, these observations suggest that RALF peptides impact on acidification and cell expansion during growth and development. Although few components of the signalling pathway have been revealed, the recent identification of FERONIA (FER) as a RALF receptor and plasma membrane H(+)-ATPase 2 as a downstream target provide a major step forward.

A fungal pathogen secretes plant alkalinizing peptides to increase infection

Article

Apr 2016

Plant infections caused by fungi are often associated with an increase in the pH of the surrounding host tissue. Extracellular alkalinization is thought to contribute to fungal pathogenesis, but the underlying mechanisms are poorly understood. Here, we show that the root-infecting fungus Fusarium oxysporum uses a functional homologue of the plant regulatory peptide RALF (rapid alkalinization factor) to induce alkalinization and cause disease in plants. An upshift in extracellular pH promotes infectious growth of Fusarium by stimulating phosphorylation of a conserved mitogen-activated protein kinase essential for pathogenicity. Fungal mutants lacking a functional Fusarium (F)-RALF peptide failed to induce host alkalinization and showed markedly reduced virulence in tomato plants, while eliciting a strong host immune response. Arabidopsis plants lacking the receptor-like kinase FERONIA, which mediates the RALF-triggered alkalinization response, displayed enhanced resistance against Fusarium. RALF homologues are found across a number of phylogenetically distant groups of fungi, many of which infect plants. We propose that fungal pathogens use functional homologues of alkalinizing peptides found in their host plants to increase their infectious potential and suppress host immunity.

A technique for studying host resistance and pathogenicity in tomato Fusarium wilt

Article

Jan 1939
PHYTOPATHOLOGY

F.L. Wellman

Phytopathogen emergence in the genomics era

Article

Feb 2015

Phytopathogens are a global threat to plant agriculture and biodiversity. The genomics era has lead to an exponential rise in comparative gene and genome studies of both economically significant and insignificant microorganisms. In this review we highlight some recent comparisons and discuss how they identify shared genes or genomic regions associated with host virulence. The two major mechanisms of rapid genome adaptation - horizontal gene transfer and hybridisation - are reviewed and we consider how intra-specific pan-genome sequences encode alternative host specificity. We also discuss the power that access to expansive gene databases provides in aiding the study of phytopathogen emergence. These databases can rapidly enable the identification of an unknown pathogen and its origin, as well as genomic adaptations required for emergence. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

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