Shigang 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.

Understanding resistant germplasm-induced virulence variation through analysis of proteomics and suppression subtractive hybridization in a maize pathogen Curvularia lunata.

Curvularia lunata is an important pathogen causing Curvularia leaf spot in maize. Significant pathogenic variation has been found in C. lunata. To better understand the mechanism of this phenomenon, we consecutively put the selective pressures of resistant maize population on C. lunata strain WS18 (low virulence) artificially. As a result, the virulence of this strain was significantly enhanced. Using 2DE, 12 up‐regulated and four down‐regulated proteins were identified in virulence‐increased strain compared to WS18. Our analysis revealed that melanin synthesis‐related proteins (Brn1, Brn2, and scytalone dehydratase) and stress tolerance‐related proteins (HSP 70) directly involved in the potential virulence growth as crucial markers or factors in C. lunata. To validate 2DE results and screen differential genes at mRNA level, we constructed a subtracted cDNA library (tester: virulence‐increased strain; driver: WS18). A total of 188 unigenes were obtained this way, of which 14 were indicators for the evolution of pathogen virulence. Brn1 and hsp genes exhibited similar expression patterns corresponding to proteins detected by 2DE. Overall, our results indicated that differential proteins or genes, being involved with melanin synthesis or tolerance response to stress, could be considered as hallmarks of virulence increase in C. lunata.

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.

Identification of a Novel Fungus, Leptosphaerulina chartarum SJTU59 and Characterization of Its Xylanolytic Enzymes

Xylanolytic enzymes are widely used in processing industries, e.g., pulp and paper, food, livestock feeds, and textile. Furthermore, certain xylanotic enzymes have demonstrated the capability to improve the resistance and immunity of plants. Screening of high-yield microbial xylanolytic enzyme producers is significant for improving large-scale cost-effective xylanolytic enzyme production. This study provided new evidence of high-level xylanolytic enzyme production by a novel fungus, designated Leptosphaerulina chartarum SJTU59. Under laboratory conditions, L. chartarum SJTU59 produced xylanolytic enzymes of up to 17.566 U/mL (i.e., 878.307 U/g substrate). The enzyme solution was relatively stable over a wide range of pH (pH 3.0 to pH 9.0) and temperature (40°C to 65°C) while showing high resistance to the majority of metal ions tested. Composition analysis of the hydrolytic products of xylan showed sufficient degradation by xylanolytic enzymes from L. chartarum SJTU59, mainly the monosaccharide xylose, and a small amount of xylobiose were enzymatically produced; whereas in the presence of sufficient xylan substrates, mainly xylooligosaccharides, an emerging prebiotic used in food industry, were produced. In addition, the xylanolytic enzyme preparation from L. chartarum SJTU59 could initiate tissue necrosis and oxidative burst in tobacco leaves, which may be related to enhanced plant defense to adversity and disease. L. chartarum SJTU59 possessed a complex xylanolytic enzyme system, from which two novel endo-β-1,4-xylanases of the glycoside hydrolase (GH) family 10, one novel endo-β-1,4-xylanase of the GH family 11, and one novel β-xylosidase of the GH family 43 were obtained via rapid amplification of complementary DNA ends. Given the high yield and stable properties of xylanolytic enzymes produced by L. chartarum SJTU59, future studies will be conducted to characterize the properties of individual xylanolytic enzymes from L. chartarum SJTU59. xylanolytic enzymes-encoding gene(s) of potential use for industrial and agricultural applications will be screened to construct genetically engineered strains.

Tmac1, a transcription factor which regulated high affinity copper transport in Trichoderma reesei

The Mac1 protein was a transcriptional activator that sensed very low concentration of copper and regulated the copper transport in Saccharomyces cerevisiae. Here, we cloned a gene from Trichoderma reesei named Tmac1, whose deduced amino acid sequence showed 29% identical to Mac1p. Furthermore, two Cys-His repeats metal binding motifs of Tmac1p, one in the 354-369C terminus and one in the 475-490C terminus were also present in Mac1p. A deletion mutant of Tmac1 was hypersensitive to the copper starvation and showed poor growth. Subsequently, the function was recovered by the gene complementation experiment. Furthermore, the Tmac1 gene fully complemented growth defects of yeast ΔMac1 mutant. The expression of Tmac1p was activated at low concentration of copper and depressed when the concentration of copper excess 1mM. Furthermore, the fluorescence intensity enhanced at copper starvation and decreased under copper excess by fusion the eGFP to the Tmac1p. It proved that the expression of Mac1p was exactly regulated by copper concentration, because eGFP and Mac1p were expressed under the control of the same one promoter. We also cloned a gene named Tctr3 with bioinformatics. With a series of experiments, we proved it was the target gene of Tmac1. To sum up, Tmac1 may encode a transcriptional activator regulated high-affinity copper transport in T. reesei.

Increased virulence of transgenic Trichoderma koningi strains to the Asian corn borer larvae by overexpressing heterologous chit42 gene with chitin-binding domains

The chit42 gene cloned from Metarhizium anisopliae lacks chitin-binding domain (chBD), which plays important roles in binding insoluble chitin. Five kinds of hybrid chitinase Trichoderma transformants were constructed in this study, where the chit42 gene was fused to chBDs derived from plant, bacterial, and insect sources. The transformant Mc4 harboring chBDs from bitter melon (Momordica charantia) displayed the highest chitinase activity among all chBDs. The chitinase activities of Mc4, chit42 Trichoderma transformant Mchit3, and wild-type strain T30 were 44.94, 32.48, and 12.38 U/mL, respectively. The mortality rate of corn borer larvae in Mc4 fermentation liquid treatment increased by 10% and 30% compared with Mchit3 and T30, respectively. The midgut microvilli and goblet cell microvilli of the corn borer larvae exhibited distinct pathological changes after 48 h of feeding in Mc4 treatment. Mc4 also exhibited the strongest antifungal activity against Fusarium verticillioides and Rhizoctonia solani.

Expression of green fluorescent protein in Curvularia lunata causing maize leaf spot

Abstract Curvularia lunata is a widespread fungal pathogen infecting maize leaves. In this study, we developed a genetic transformation system for C. lunata by using the plasmid (pCPXHY1Egfp) with both enhanced green fluorescent protein gene egfp and hygromycin resistance gene hph. GFP rendered hyphae of the transformants fluorescent in culture. Most transformants that showed constitutively expressing EGFP were comparable to the wild-type strain in morphological features. PCR and Southern blot analyses of the hph gene further confirmed that the plasmid was integrated into the genome of C. lunata. This is the first report that a ‘reporter’ gene, egfp, could be stably expressed in C. lunata. Using the transformants with EGFP marker will assist us to visualize the features of infection at different stages.

Impact on bacterial community in midguts of the Asian corn borer larvae by transgenic Trichoderma strain overexpressing a heterologous chit42 gene with chitin-binding domain.

This paper is the first report of the impact on the bacterial community in the midgut of the Asian corn borer (Ostrinia furnacalis) by the chitinase from the transgenic Trichoderma strain. In this study, we detected a change of the bacterial community in the midgut of the fourth instar larvae by using a culture-independent method. Results suggested that Proteobacteria and Firmicutes were the most highly represented phyla, being present in all the midgut bacterial communities. The observed species richness was simple, ranging from four to five of all the 16S rRNA clone libraries. When using Trichoderma fermentation liquids as additives, the percentages of the dominant flora in the total bacterial community in larval midgut changed significantly. The community of the genus Ochrobactrum in the midgut decreased significantly when the larvae were fed with the fermentation liquids of the transgenic Trichoderma strain Mc4. However, the Enterococcus community increased and then occupied the vacated niche of the Ochrobactrum members. Furthermore, the Shannon–Wiener (H) and the Simpson (1-D) indexes of the larval midgut bacterial library treated by feeding fermentation liquids of the transgenic Trichoderma strain Mc4 was the lowest compared with the culture medium, fermentation liquids of the wild type strain T30, and the sterile artificial diet. The Enterococcus sp. strain was isolated and characterized from the healthy larvae midgut of the Asian corn borer. An infection study of the Asian corn borer larvae using Enterococcus sp. ACB-1 revealed that a correlation existed between the increased Enterococcus community in the larval midgut and larval mortality. These results demonstrated that the transgenic Trichoderma strain could affect the composition of the midgut bacterial community. The change of the midgut bacterial community might be viewed as one of the factors resulting in the increased mortality of the Asian corn borer larvae.

Impacts on silkworm larvae midgut proteomics by transgenic Trichoderma strain and analysis of glutathione S-transferase sigma 2 gene essential for anti-stress response of silkworm larvae

Lepidoptera is a large order of insects that have major impacts on humans as agriculture pests. The midgut is considered an important target for insect control. In the present study, 10 up-regulated, 18 down-regulated, and one newly emerged protein were identified in the transgenic Trichoderma-treated midgut proteome. Proteins related to stress response, biosynthetic process, and metabolism process were further characterized through quantitative real-time PCR (qPCR). Of all the identified proteins, the glutathione S-transferase sigma 2 (GSTs2) gene displayed enhanced expression when larvae were fed with Trichoderma wild-type or transgenic strains. Down regulation of GSTs2 expression by RNA interference (RNAi) resulted in inhibition of silkworm growth when larvae were fed with mulberry leaves treated with the transgenic Trichoderma strain. Weight per larva decreased by 18.2%, 11.9%, and 10.7% in the untreated control, ddH2O, and GFP dsRNA groups, respectively, at 24h, while the weight decrease was higher at 42.4%, 28.8% and 32.4% at 72 h after treatment. Expression of glutathione S-transferase omega 2 (GSTo2) was also enhanced when larvae were fed with mulberry leaves treated with the transgenic Trichoderma strain. These results indicated that there was indeed correlation between enhanced expression of GSTs2 and the anti-stress response of silkworm larvae against Trichoderma. This study represents the first attempt at understanding the effects of transgenic organisms on the midgut proteomic changes in silkworm larvae. Our findings could not only broaden the biological control targets of insect at the molecular level, but also provide a theoretical foundation for biological safety evaluation of the transgenic Trichoderma strain.

Cloning and function analysis of a drought-inducible gene associated with resistance to Curvularia leaf spot in maize

ZmDIP was cloned and its function against Curvularia lunata was analyzed, according to a previous finding on a drought-inducible protein in resistant maize identified through MALDI-TOF-MS/MS. The ZmDIP expression varied in roots, leaf sheaths, and young, as well as old, leaves of different maize inbred lines. The ZmDIP transcript level changed in leaves over the course of time after inoculation with C.lunata. A prokaryotic expression analysis demonstrated that the gene can regulate the salt stress tolerance of Escherichia coli. The ZmDIP transient expression in the maize leaf showed that the gene was also linked to leaf resistance against the C.lunata infection. ZmDIP-mediated ROS and ABA signaling pathways were inferred to be closely associated with maize leaf resistance to the pathogen infection.