Background: WRKY proteins are a large family of transcriptional regulators in higher plant. They are involved in many biological processes, such as plant development, metabolism, and responses to biotic and abiotic stresses. Prior to the present study, only one full-length cucumber WRKY protein had been reported. The recent publication of the draft genome sequence of cucumber allowed us to conduct a genome-wide search for cucumber WRKY proteins, and to compare these positively identified proteins with their homologs in model plants, such asArabidopsis.Results: We identified a total of 55 WRKY genes in the cucumber genome. According to structural features of their encoded proteins, the cucumber WRKY (CsWRKY) genes were classified into three groups (group 1-3). Analysis of expression profiles ofCsWRKYgenes indicated that 48 WRKY genes display differential expression either in their transcript abundance or in their expression patterns under normal growth conditions, and 23 WRKY genes were differentially expressed in response to at least one abiotic stresses (cold, drought or salinity). The expression profile of stress-inducibleCsWRKYgenes were correlated with those of their putativeArabidopsis WRKY (AtWRKY)orthologs, except for the group 3 WRKY genes. Interestingly, duplicated group 3AtWRKYgenes appear to have been under positive selection pressure during evolution. In contrast, there was no evidence of recent gene duplication or positive selection pressure amongCsWRKYgroup 3 genes, which may have led to the expressional divergence of group 3 orthologs.Conclusions: Fifty-five WRKY genes were identified in cucumber and the structure of their encoded proteins, their expression, and their evolution were examined. Considering that there has been extensive expansion of group 3 WRKY genes in angiosperms, the occurrence of different evolutionary events could explain the functional divergence of these genes.

Losses in crops caused by plant pathogenic bacteria and parasitic nematode are increasing because of a decrease in efficacy of traditional management measures. There is an urgent need to develop nonchemical and ecofriendly based management to control plant diseases. A potential approach of controlling plant disease in the crops is the use of biocontrol agents and their secondary metabolites (SMs). Luckily fungi and especially the genus Trichoderma comprise a great number of fungal strains that are the potential producer of bioactive secondary metabolites. In this study secondary metabolites from ten Trichoderma spp. were evaluated for their antibacterial and nematicidal potential against phytopathogenic bacteria Ralstonia solanacearum, Xanthomonas compestris and plant parasitic nematode Meloidogyne incognita. Five different growth media were evaluated for the production of SMs. It was shown that SMs of different Trichoderma spp. obtained on different growth media were different in the degree of their bioactivity. Comparison of five growth media showed that SMs produced on solid wheat and STP media gave higher antibacterial activity. SMs of T. pseudoharzianum (T113) obtained on solid wheat media were more effective against the studied bacteria followed by SMs from T. asperelloides (T136), T. pseudoharzianum (T129) and T. pseudoharzianum (T160). Scanning electron microscopy (SEM) was further conducted to observe the effect of SMs on bacterial cell morphology. As evident from the SEM, SMs produced severe morphological changes, such as rupturing of the bacterial cell walls, disintegration of cell membrane and cell content leaking out. SMs from T. viridae obtained on liquid STP and solid wheat media showed the highest percent of M. incognita juveniles (J2s) mortality and inhibition in egg hatching of M. incognita. The results of our study suggest that T. pseudoharzianum (T113) and T. viridae could be selected as an effective candidate for SMs source against phytopathogenic bacteria and M. incognita respectively.

The fungus Pochonia chlamydosporia parasitizes nematode eggs and has become one of the most promising biological control agents (BCAs) for plant-parasitic nematodes, which are major agricultural pests that cause tremendous economic losses worldwide. The complete mitochondrial (mt) genome is expected to open new avenues for understanding the phylogenetic relationships and evolution of the invertebrate-pathogenic fungi in Hypocreales. The complete mitogenome sequence of P. chlamydosporia is 25,615 bp in size, containing the 14 typical protein-coding genes, two ribosomal RNA genes, an intronic ORF coding for a putative ribosomal protein (rps3) and a set of 23 transfer RNA genes (trn) which recognize codons for all amino acids. Sequence similarity studies and syntenic gene analyses show that 87.02% and 58.72% of P. chlamydosporia mitogenome sequences match 90.50% of Metarhizium anisopliae sequences and 61.33% of Lecanicillium muscarium sequences with 92.38% and 86.04% identities, respectively. A phylogenetic tree inferred from 14 mt proteins in Pezizomycotina fungi supports that P. chlamydosporia is most closely related to the entomopathogenic fungus M. anisopliae. The invertebrate-pathogenic fungi in Hypocreales cluster together and clearly separate from a cluster comprising plant-pathogenic fungi (Fusarium spp.) and Hypocrea jecorina. A comparison of mitogenome sizes shows that the length of the intergenic regions or the intronic regions is the major size contributor in most of mitogenomes in Sordariomycetes. Evolutionary analysis shows that rps3 is under positive selection, leading to the display of unique evolutionary characteristics in Hypocreales. Moreover, the variability of trn distribution has a clear impact on gene order in mitogenomes. Gene rearrangement analysis shows that operation of transposition drives the rearrangement events in Pezizomycotina, and most events involve in trn position changes, but no rearrangement was found in Clavicipitaceae. We present the complete annotated mitogenome sequence of P. chlamydosporia. Based on evolutionary and phylogenetic analyses, we have determined the relationships between the invertebrate-pathogenic fungi in Hypocreales. The invertebrate-pathogenic fungi in Hypocreales referred to in this paper form a monophyletic group sharing a most recent common ancestor. Our rps3 and trn gene order results also establish a foundation for further exploration of the evolutionary trajectory of the fungi in Hypocreales.

Peptidases are very important to parasites, which have central roles in parasite biology and pathogenesis. In this study, by comparative genome analysis, genome-wide peptidase diversities among plant-parasitic nematodes are estimated. We find that genes encoding cysteine peptidases in family C13 (legumain) are significantly abundant in pine wood nematodes Bursaphelenchus genomes, compared to those in other plant-parasitic nematodes. By phylogenetic analysis, a clade of B. xylophilus-specific legumain is identified. RT-qPCR detection shows that these genes are highly expressed at early stage during the nematode infection process. Utilizing transgene technology, cDNAs of three species-specific legumain were introduced into the Arabidopsis γvpe mutant. Functional complementation assay shows that these B. xylophilus legumains can fully complement the activity of Arabidopsis γVPE to mediate plant cell death triggered by the fungal toxin FB1. Secretory activities of these legumains are experimentally validated. By comparative transcriptome analysis, genes involved in plant cell death mediated by legumains are identified, which enrich in GO terms related to ubiquitin protein transferase activity in category molecular function, and response to stimuli in category biological process. Our results suggest that B. xylophilu-specific legumains have potential as effectors to be involved in nematode-plant interaction and can be related to host cell death.