Role of two secreted proteins from Trichoderma virens in mycoparasitism and induction of plant resistance

The soil-borne filamentous fungus Trichoderma virens is a biocontrol agent with a well known ability to produce antibiotics, parasitize pathogenic fungi and induce systemic resistance in plants. Here we report the identification, purification and characterization of an elicitor secreted by T. virens; a small protein designated Sm1 (small protein 1). Confrontation and disk assays demonstrated that Sm1 lacks toxic activity against plants and microbes. Native, purified Sm1 triggers production of reactive oxygen species in rice (Oryza sativa) and cotton (Gossypium hirsutum), and induces the expression of defense related genes both locally and systemically in cotton. Gene expression analysis revealed that SM1 is expressed throughout fungal development and is transcriptionally regulated by nutrient conditions and the presence of a host plant. When T. virens was co-cultured with cotton in an axenic hydroponic system, SM1 expression and secretion of the protein was significantly higher than when the fungus was grown alone. These results indicate that Sm1 is involved in plant-Trichoderma recognition and the induction of resistance by activation of plant defense mechanisms. Following the cloning of SM1, strains disrupted in or over-expressing SM1 were generated. Targeted gene disruption revealed that SM1 was not involved in fungal development. Expression of defense related genes in cotton and maize (Zea mays) was induced locally and systemically following colonization by T. virens in the hydroponic system. Low levels of expression of cotton or maize defense genes were found when seedlings were grown with a T. virens strain disrupted in SM1, ssupporting the Sm1-elicitor hypothesis. Additionally, unique proteins in T.virens-cotton/maize interaction were identified. Thus, the induction of defense responses in two agriculturally important crops appears to be microbially mediated. Functional analysis of a cell wall degrading enzyme, beta-1,6-glucananse (Tv-bgn3) from T. virens, demonstrated involvement of this enzyme indirectly in mycoparasitic activity of T. virens. Protein extracts from the strain disrupted in TV-BGN3 displayed reduced capability to inhibit growth of Pythium ultimum as compared to the wild-type. Additionally, protein extracts from the strains co-expressed with TV-BGN2 (beta-1,3-glucananse) from T. virens showed a significantly increased capability to inhibit growth of P. ultimum and Rhizoctonia solani hyphae

Genome-Wide Identification of Pseudomonas aeruginosa Virulence-Related Genes Using a Caenorhabditis elegans Infection Model

Pseudomonas aeruginosa strain PA14 is an opportunistic human pathogen capable of infecting a wide range of organisms including the nematode Caenorhabditis elegans. We used a non-redundant transposon mutant library consisting of 5,850 clones corresponding to 75% of the total and approximately 80% of the non-essential PA14 ORFs to carry out a genome-wide screen for attenuation of PA14 virulence in C. elegans. We defined a functionally diverse 180 mutant set (representing 170 unique genes) necessary for normal levels of virulence that included both known and novel virulence factors. Seven previously uncharacterized virulence genes (ABC transporters PchH and PchI, aminopeptidase PepP, ATPase/molecular chaperone ClpA, cold shock domain protein PA0456, putative enoyl-CoA hydratase/isomerase PA0745, and putative transcriptional regulator PA14_27700) were characterized with respect to pigment production and motility and all but one of these mutants exhibited pleiotropic defects in addition to their avirulent phenotype. We examined the collection of genes required for normal levels of PA14 virulence with respect to occurrence in P. aeruginosa strain-specific genomic regions, location on putative and known genomic islands, and phylogenetic distribution across prokaryotes. Genes predominantly contributing to virulence in C. elegans showed neither a bias for strain-specific regions of the P. aeruginosa genome nor for putatively horizontally transferred genomic islands. Instead, within the collection of virulence-related PA14 genes, there was an overrepresentation of genes with a broad phylogenetic distribution that also occur with high frequency in many prokaryotic clades, suggesting that in aggregate the genes required for PA14 virulence in C. elegans are biased towards evolutionarily conserved genes

<i>P. aeruginosa</i> PA14 virulence-attenuated genes identified in the <i>C. elegans</i> infection model.

<p>Genes are listed in descending order of contribution to virulence (according to the ratio of mutant LT₅₀/wild-type LT₅₀) using the data from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002813#ppat-1002813-g002" target="_blank">Figure 2</a>. Genes previously identified as required for normal levels of <i>P. aeruginosa</i> virulence in various model systems are indicated. In some cases only <i>P. aeruginosa</i> strains other than PA14 were examined and the strain and mode of killing is indicated in parentheses. The other pathogens, in which orthologs of these genes have been implicated in virulence, are noted in the last column.</p

Among the PA14 genes required for virulence in <i>C. elegans</i>, “<i>Pseudomonas</i>-genus-specific” (PGS) genes are underrepresented, whereas “high-frequency-broad-phylogeny” (HFBP) genes are overrepresented.

<p>Based on phylostratigraphic analysis, PA14 genes required for virulence in <i>C. elegans</i> were classified as either “<i>Pseudomonas</i>-genus-specific” (PGS), presumably representing the newest genes in PA14, “high-frequency-broad-phylogeny” (HFBP), representing the oldest, most conserved genes in PA14, or “all others”. The percentage of each gene set, including the PA14 genome genes, the PA14-NR, primary, secondary, tertiary, auxotroph, and VFDB gene sets that are classified as PGS genes, HFBP genes, or all others genes, are shown. HFBP genes comprise 10% of the PA14 genome, and about 7% of the NR set genes. Furthermore, HFBP genes are increasingly overrepresented with successive iterations of the screen accounting for 13% of the primary set (p-value = 0.00004), 14% of the secondary set (p-value = 0.0005) and 19% of the tertiary set (p-value = 0.006). HFBP genes make up greater than 50% of the auxotroph set with a (p-value = 5.47×10⁻²⁸) relative to the NR set. The PA14 VFDB set contains an underrepresentation of HFBP genes (1.6%, p-value = 0.0001). PGS genes make up 11% and 9.6% of the PA14 genome and NR set respectively. Over successive iterations of the screen, PGS genes become numerically more underrepresented relative to the NR set, comprising 5.7% of the primary set (5.7%, p-value = 0.01), 5.2% of the secondary set (p-value = 0.03, not statistically significant), and 2.4% of the tertiary set (p-value = 0.08, not statistically significant). Due to the small numbers of genes in the secondary and tertiary sets, only the underrepresentation in the primary set is significant after application of multiple comparison correction (FDR, q< = 0.05). PGS genes are underrepresented in the auxotroph set (0%, p-value = 0.0006). Statistical data for this figure are presented in supplemental <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002813#ppat.1002813.s020" target="_blank">Table S7</a>.</p

Pipeline of screen for PA14 virulence-attenuated mutants in <i>C. elegans</i>.

<p>The three screening steps for identification of <i>P. aeruginosa</i> PA14 virulence-attenuated mutants are outlined; details of the screens are presented in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002813#s4" target="_blank">Materials and Methods</a> and the text. The number of mutants obtained after each round of screening, as well as those removed from the pool for various reasons, is shown. Note that the 313 mutants identified in the primary screen and the 180 from the secondary screen represent 294 and 170 unique genes respectively because some genes were represented by multiple mutants, and a small fraction of mutants were in intergenic regions (see text). In the tertiary screen a single mutant defined each gene.</p

41 PA14 genes required for virulence in a <i>C. elegans</i> infection based killing model.

<p>The ratio of nematode survival on mutant PA14 to that on wild-type PA14 (mutant LT₅₀/WT LT₅₀) is presented for 41 mutants identified after three rounds of screening as well as for the known virulence-attenuated mutants, <i>lasR</i> and <i>pilA</i>. The time to 50% death (LT₅₀) was calculated using a non-linear regression based on the Hill equation (Prism 5.0). 100–150 animals were tested in each experiment. Error bars represent the SEM of the ratios derived from at least two different experiments (lack of error bars indicates that the mutants for known virulence factors <i>gacA</i>, <i>ptsP</i> and <i>vfr</i> were tested only once). Red bars depict the ratio of the LT₅₀ of <i>lasR</i> or <i>pilA</i> to WT PA14. The <i>lasR</i> and <i>pilA</i> mutants were generated previously (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002813#s4" target="_blank">Materials and Methods</a>); there are no alleles of <i>lasR</i> or <i>pilA</i> in the NR library. The number of alleles tested with an avirulent phenotype is indicated by a number below the graph: 1 indicates that a single allele was tested but that there exist multiple alleles in the master transposon library, S indicates only a single allele was available in the library. Genes that are predicted to be in operons are indicated (Y = yes, N = no). Genes in a single operon are represented in the same color and an underline designates that other genes within the same operon were tested for their role in virulence.</p

The catabolic arginine succinyltransferase (<i>aru</i>) operon is required for normal virulence in <i>C. elegans</i>.

<p>A) <i>aruFGDB</i> is transcribed as a unit; the transcriptional regulator <i>aruC</i> is transcribed separately. The <i>aruFGDB</i> operon encodes enzymes for the major aerobic route of arginine utilization as an energy, carbon and nitrogen source <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002813#ppat.1002813-Schneider1" target="_blank">[56]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002813#ppat.1002813-Itoh1" target="_blank">[57]</a>. In <i>P. aeruginosa</i> PA01, <i>aruE</i> belongs to a separate transcription unit <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002813#ppat.1002813-Itoh1" target="_blank">[57]</a>. B) <i>MAR2xT7</i> insertions in <i>aruC</i>, <i>aruF</i>, <i>aruG</i>, <i>aruD</i> and <i>aruB</i> all reduce the virulence of PA14. The single mutation in <i>aruE</i> has normal virulence.</p

The distribution of <i>P. aeruginosa</i> PA14 genes required for virulence in <i>C. elegans</i> in the Core vs. Auxiliary genome and on both predicted and known genomic islands.

<p>A) The percentages of <i>P. aeruginosa</i> PA14 genomic genes, PA14-NR Set mutants, and primary, secondary, tertiary, auxotroph, and VFDB set genes that are part of the <i>P. aeruginosa</i> core and auxiliary genome as defined by Mathee et al. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002813#ppat.1002813-Mathee1" target="_blank">[41]</a>. The auxotroph set is disproportionately part of the core genome. Genes in the primary, secondary, tertiary, and VFDB sets have proportions in the core and auxiliary genes that are statistically indistinguishable from the PA14 NR set and from the genome as a whole. B) The percentages of genes from the PA14 genome, the PA14-NR Set, and from the primary, secondary, tertiary, auxotroph, and VFDB sets that are located on genomic islands predicted by IslandViewer. Representation of primary, secondary, and tertiary gene sets on predicted islands was statistically representative of the genome as a whole and of the NR-set, whereas VFDB genes had a statistical overrepresentation of genes located on predicted genomic islands (p = 0.0005). C) The percentages of genes from the PA14 genomic, the PA14-NR Set, and from the primary, secondary, tertiary, auxotroph, and VFDB sets located on the known PAPI-1, PAPI-2, and PAGI-1 genomic islands. Representation of primary, secondary, and tertiary set genes was statistically identical to the genome as a whole and the NR-set. VFDB genes were statistically underrepresented on the known islands (p = 0.007). Refer to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002813#ppat.1002813.s020" target="_blank">Table S7</a> for statistics.</p

<i>ΔexoU</i> may be a sensitized background that can reveal virulence-associated genes.

<p>Deletion of the Type III effector protein ExoU has no statistically significant impact on PA14 virulence in <i>C. elegans</i> (B and D and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002813#ppat.1002813-Miyata1" target="_blank">[25]</a>). A) Hypothetical protein PA4016 and adjacent loci PA4017 and PA4015; PA4016 is most likely a single gene transcription unit. B) A PA4016 <i>MAR2xT7</i> insertion mutant (#22683) in the <i>ΔexoU</i> strain background has attenuated virulence in <i>C. elegans</i>, but a second PA4016 insertion allele (#41856) in the WT strain does not. C) Glutathione synthetase, <i>gshB</i> (PA0407), is a single gene transcription unit. D) Multiple alleles of <i>gshB</i> exhibit reduced virulence in <i>C. elegans</i> but the <i>gshB</i> #6613 allele in the <i>ΔexoU</i> strain background is more attenuated.</p