Jinxin Gao

Genome sequence and virulence variation-related transcriptome profiles of Curvularia lunata, an important maize pathogenic fungus

BackgroundCurvularia lunata is an important maize foliar fungal pathogen that distributes widely in maize growing area in China. Genome sequencing of the pathogen will provide important information for globally understanding its virulence mechanism.ResultsWe report the genome sequences of a highly virulent C. lunata strain. Phylogenomic analysis indicates that C. lunata was evolved from Bipolaris maydis (Cochliobolus heterostrophus). The highly virulent strain has a high potential to evolve into other pathogenic stains based on analyses on transposases and repeat-induced point mutations. C. lunata has a smaller proportion of secreted proteins as well as B. maydis than entomopathogenic fungi. C. lunata and B. maydis have a similar proportion of protein-encoding genes highly homologous to experimentally proven pathogenic genes from pathogen-host interaction database. However, relative to B. maydis, C. lunata possesses not only many expanded protein families including MFS transporters, G-protein coupled receptors, protein kinases and proteases for transport, signal transduction or degradation, but also many contracted families including cytochrome P450, lipases, glycoside hydrolases and polyketide synthases for detoxification, hydrolysis or secondary metabolites biosynthesis, which are expected to be crucial for the fungal survival in varied stress environments. Comparative transcriptome analysis between a lowly virulent C. lunata strain and its virulence-increased variant induced by resistant host selection reveals that the virulence increase of the pathogen is related to pathways of toxin and melanin biosynthesis in stress environments, and that the two pathways probably have some overlaps.ConclusionsThe data will facilitate a full revelation of pathogenic mechanism and a better understanding of virulence differentiation of C. lunata.

Insertional Mutagenesis and Cloning of the Gene Required for the Biosynthesis of the Non-Host-Specific Toxin in Cochliobolus lunatus that Causes Maize Leaf Spot

The maize pathotype Cochliobolus lunatus causes Curvularia leaf spot by producing a non-host-specific toxin known as methyl 5-(hydroxymethyl) furan-2-carboxylate (M5HF2C). However, related research that explores the genes that control the production of this toxin is rare. In the current work, Agrobacterium tumefaciens-mediated transformation (ATMT) was employed to tag the gene required for the biosynthesis of the toxin. Of the 3,000 ATMT transformants recovered, 4 showed a significant decline in pathogenicity on maize leaves; 1 transformant, T806, produced no detectable M5HF2C. Genomic DNA that flanks the integrated plasmid was recovered from one of the mutants. A cosmid clone of the wild-type strain was isolated using the recovered DNA as a probe. The results of the structural and functional analyses of the region corresponding to the tagged site were then used as a basis to successfully clone one gene, called Clt-1. Bioinformatics analysis showed that the gene coded a BTB domain-containing protein that comprises 745 amino acids. Southern analysis revealed that the gene was localized in the genome as a single copy. The essential roles of Clt-1 in both toxin production and pathogenicity were confirmed by gene disruption experiments. In summary, the novel gene Clt-1 is closely associated with toxin production and pathogen virulence in leaves of susceptible varieties.

Biological role of Trichoderma harzianum-derived platelet-activating factor acetylhydrolase (PAF-AH) on stress response and antagonism.

We investigated the properties of platelet-activating factor acetylhydrolase (PAF-AH) derived from Trichoderma harzianum. The enzyme, comprised of 572 amino acids, shares high homology with PAF-AH proteins from T. koningii and other microbial species. The optimum enzymatic activity of PAF-AH occurred at pH 6 in the absence of Ca2+ and it localized in the cytoplasm, and we observed the upregulation of PAF-AH expression in response to carbon starvation and strong heat shock. Furthermore, PAF-AH knockout transformant growth occurred more slowly than wild type cells and over-expression strains grown in SM medium at 37°C and 42°C. In addition, PAF-AH expression significantly increased under a series of maize root induction assay. Eicosanoic acid and ergosterol levels decreased in the PAF-AH knockouts compared to wild type cells, as revealed by GC/MS analysis. We also determined stress responses mediated by PAF-AH were related to proteins HEX1, Cu/Zn superoxide dismutase, and cytochrome c. Finally, PAF-AH exhibited antagonistic activity against Rhizoctonia solani in plate confrontation assays. Our results indicate PAF-AH may play an important role in T. harzianum stress response and antagonism under diverse environmental conditions.

Effect of Trichoderma harzianum on maize rhizosphere microbiome and biocontrol of Fusarium Stalk rot

Fusarium stalk rot (FSR) caused by Fusarium graminearum (FG) significantly affects the productivity of maize grain crops. Application of agrochemicals to control the disease is harmful to environment. In this regard, use of biocontrol agent (BCA) is an alternative to agrochemicals. Although Trichoderma species are known as BCA, the selection of host-pathogen specific Trichoderma is essential for the successful field application. Hence, we screened a total of 100 Trichoderma isolates against FG, selected Trichoderma harzianum (CCTCC-RW0024) for greenhouse experiments and studied its effect on changes of maize rhizosphere microbiome and biocontrol of FSR. The strain CCTCC-RW0024 displayed high antagonistic activity (96.30%), disease reduction (86.66%), biocontrol-related enzyme and gene expression. The root colonization of the strain was confirmed by eGFP tagging and qRT-PCR analysis. Pyrosequencing revealed that exogenous inoculation of the strain in maize rhizosphere increased the plant growth promoting acidobacteria (18.4%), decreased 66% of FG, and also increased the plant growth. In addition, metabolites of this strain could interact with pathogenicity related transcriptional cofactor FgSWi6, thereby contributing to its inhibition. It is concluded that T. harzianum strain CCTCC-RW0024 is a potential BCA against FSR.

Involvement of a velvet protein ClVelB in the regulation of vegetative differentiation, oxidative stress response, secondary metabolism, and virulence in Curvularia lunata

The ortholog of Aspergillus nidulans VelB, which is known as ClVelB, was studied to gain a broader insight into the functions of a velvet protein in Curvularia lunata. With the expected common and specific functions of ClVelB, the deletion of clvelB results in similar though not identical phenotypes. The pathogenicity assays revealed that ΔClVelB was impaired in colonizing the host tissue, which corresponds to the finding that ClVelB controls the production of conidia and the methyl 5-(hydroxymethyl) furan-2-carboxylate toxin in C. lunata. However, the deletion of clvelB led to the increase in aerial hyphae and melanin formation. In addition, ΔClVelB showed a decreased sensitivity to iprodione and fludioxonil fungicides and a decreased resistance to cell wall-damaging agents and osmotic stress and tolerance to H2O2. The ultrastructural analysis indicated that the cell wall of ΔClVelB became thinner, which agrees with the finding that the accumulated level of glycerol in ΔClVelB is lower than the wild-type. Furthermore, the interaction of ClVelB with ClVeA and ClVosA was identified in the present research through the yeast two-hybrid and bimolecular fluorescence complementation assays. Results indicate that ClVelB plays a vital role in the regulation of various cellular processes in C. lunata.

Identification of proteins associated with the production of melanin and with pathogenicity in maize pathogen Curvularia lunata

Curvularia lunata produces 1,8-dihydroxynaphthalene (DHN) melanin. Previously, we have successfully cloned and silenced the Brn1 gene, which is involved in DNH melanin production and pathogenicity. However, we hypothesise that other proteins may also be related to melanin production or pathogenicity. In this study, a proteomic approach based on two-dimensional gel electrophoresis and mass spectrometry techniques was used to identify the specific proteins associated with Brn1 expression in C. lunata wild-type CX-3 and Brn1-silenced mutant T5. One up-regulated and seven down-regulated proteins were successfully identified in T5. Whilst transaldolase, NADP-dependent mannitol dehydrogenase, formate dehydrogenase, and UTP-glucose-1-phosphate uridylyltransferase are involved in energy metabolism, elongation factor 3 facilitates the synthesis of important proteins required for the production of melanin and stress tolerance-related proteins (HSP30 and HSP70) and catalase (CAT) contribute to the pathogenicity of C. lunata. Of these proteins, CAT was further analysed as a protein of interest. Bioinformatics analysis showed that CAT consists of 748 amino acids and clustered into one branch with Dothideomycete CAT homologues in a phylogenetic tree. Further research on the exact role of the CAT gene is underway.

The platelet-activating factor acetylhydrolase gene derived from Trichoderma harzianum induces maize resistance to Curvularia lunata through the jasmonic acid signaling pathway

Platelet-activating factor acetylhydrolase (PAF-AH) derived from Trichoderma harzianum was upregulated by the interaction of T. harzianum with maize roots or the foliar pathogen Curvularia lunata. PAF-AH was associated with chitinase and cellulase expressions, but especially with chitinase, because its activity in the KO40 transformant (PAF-AH disruption transformant) was lower, compared with the wild-type strain T28. The result demonstrated that the colonization of maize roots by T. harzianum induced systemic protection of leaves inoculated with C. lunata. Such protection was associated with the expression of inducible jasmonic acid pathway-related genes. Moreover, the data from liquid chromatography-mass spectrometry confirmed that the concentration of jasmonic acid in maize leaves was associated with the expression level of defense-related genes, suggesting that PAF-AH induced resistance to the foliar pathogen. Our findings showed that PAF-AH had an important function in inducing systemic resistance to maize leaf spot pathogen.

Biological control of southern corn leaf blight by Trichoderma atroviride SG3403

Trichoderma atroviride SG3403 showed high biocontrol activity against southern corn leaf blight (SCLB; pathogen: Cochliobolus heterostrophus). T. atroviride SG3403 could cause death of C. heterostrophus race O hypha on plates. Spraying T. atroviride SG3403 conidia suspension over maize seedling leaves protected the corn from SCLB infection. Biocontrol effect lasted for 30 days in the field. Trichoderma strain was able to induce resistance response in corn leaves against pathogen infection. In corn leaves treated with T. atroviride SG3403, the enzyme activities of phenylalanine ammonia lyase (PAL) and superoxide dismutase (SOD) reached the highest at 24 h, enzyme activity of catalase (CAT) reached the highest at 36 h after inoculation of pathogen C. heterostrophus race O. RNA expression levels of Pal, Sod and Cat (which synthesis enzyme PAL, SOD and CAT) were also upregulated and corresponded to the enzyme activity at the same time point. Enzyme activities and corresponding genes expression induced by Trichoderma SG3403 was more obvious than that induced by pathogen only, which implies that T. atroviride SG3403 induced corn defense gene expression against pathogen infection. Thus, induced resistance mechanism was possibly involved in the biocontrol of SCLB by T. atroviride SG3403.

Sod gene of Curvularia lunata is associated with the virulence in maize leaf

Abstract Curvularia leaf spot, caused mainly by Curvularia lunata, is a widespread plant disease in China. In the recent years, directional host selection by the pathogen, which likely results in the virulence differentiation in pathogens, is widely reported. Among the hallmarks potentially associated to pathogen variation in virulence, superoxide dismutase gene Sod has been found to be closely related to the enhancement of virulence. In the present study, the full-length of Sod was obtained via Blastn alignment against GenBank and the whole genome of C. lunata. In order to understand the role of Sod in the virulence variation in C. lunata, targeted gene disruption was performed to construct Sod mutants. The cell wall degrading enzyme (CWDE) activities and toxin production of ΔSod were not distinctly different from wild-type strain CX-3 and its complon. However, at an early stage of infection, ΔSod virulence appeared to be lower than CX-3 and the complon, while at a later stage, its virulence gradually returned to the level of CX-3 and the complon. Furthermore, the melanin production of ΔSod was significantly reduced compared to CX-3 and the complon, suggesting that Sod gene influences the virulence by regulating melanin production at an early stage of infection but is not essential for pathogenicity. However, the disruption of Sod did not significantly affect the transcriptional expression of the melanin biosynthesis-associated genes, brn1 and scd. Therefore, we infer that Sod in C. lunata are involved, to some extent, with the virulence in maize leaf, but still needs further studies to have a clear understanding of its mechanism.

Identification of proteins associated with the production of methyl 5-(hydroxymethyl) furan-2-carboxylate toxin in Curvularia lunata

Curvularia lunata produces a non-host specific toxin known as methyl 5-(hydroxymethyl) furan-2-carboxylate (M5HF2C), a determinant of high virulence to maize. We constructed mutants through Agrobacterium tumefaciens-mediated transformation and found that mutant T806 showed a significant decline in pathogenicity on maize leaves and produced no detectable M5HF2C. Although the tagged gene (Clt-1) has been cloned and demonstrated to be closely association with M5HF2C production and pathogenicity, we expected hypothesized that other proteins might also be related to toxin production. Therefore we used two-dimensional gel electrophoresis to profile T806, and found that there were two up-regulated and six missing proteins in T806 compared to C. lunata wild-type CX-3. These results indicated that these proteins were associated with signal transduction, protein synthesis, secondary metabolism and pathogenicity. Real-time quantitative PCR analysis of five selected genes showed a correlation between low gene expression and detection of the corresponding proteins. Of these, dihydropteroate (DHFA) synthase may be more likely involved in M5HF2C biosynthesis. Bioinformatics analysis showed that DHFA synthase consists of 469 amino acids, which is clustered into one branch with dothideomycete DHFA synthase homolologs in a phylogenetic tree. Further study on the exact role of the DHFA synthase gene is underway.