The Roles of Gti1/Pac2 Family Proteins in Fungal Growth, Morphogenesis, Stress Response, and Pathogenicity.

Gti1/Pac2 is a fungal-specific transcription factor family with a stable and conserved N-terminal domain. Generally, there are two members in this family, named Gti1/Wor1/Rpy1/Mit1/Reg1/Ros1/Sge1 and Pac2, which are involved in fungal growth, development, stress response, spore production, pathogenicity, and so on. The Gti1/Pac2 family proteins share some conserved and distinct functions. For example, in Schizosaccharomyces pombe, Gti1 promotes the initiation of gluconate uptake during glucose starvation, while Pac2 controls the onset of sexual development in a pathway independent of the cAMP cascade. In the last two decades, more attention was focused on the Gti1 and its orthologs because of their significant effect on morphological switching and fungal virulence. By contrast, limited work was published on the functions of Pac2, which is required for stress responses and conidiation, but plays a minor role in fungal virulence. In this review, we present an overview of our current understanding of the Gti1/Pac2 proteins that contribute to fungal development and/or pathogenicity and of the regulation mechanisms during infection related development. Understanding the working networks of the conserved Gti1/Pac2 transcription factors in fungal pathogenicity not only advances our knowledge of the highly elaborate infection process but may also lead to the development of novel strategies for the control of plant disease. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Comparative transcriptome analysis reveals the importance of phenylpropanoid biosynthesis for the induced resistance of 84K poplar to anthracnose.

BACKGROUND: Poplar anthracnose, which is one of the most important tree diseases, is primarily caused by Colletotrichum gloeosporioides, which has been detected in poplar plantations in China and is responsible for serious economic losses. The characteristics of 84K poplar that have made it one of the typical woody model plants used for investigating stress resistance include its rapid growth, simple reproduction, and adaptability. RESULTS: In this study, we found that the resistance of 84K poplar to anthracnose varied considerably depending on how the samples were inoculated of the two seedlings in each tissue culture bottle, one (84K-Cg) was inoculated for 6 days, whereas the 84K-DCg samples were another seedling inoculated at the 6th day and incubated for another 6 days under the same conditions. It was showed that the average anthracnose spot diameter on 84K-Cg and 84K-DCg leaves was 1.23 ± 0.0577 cm and 0.67 ± 0.1154 cm, respectively. Based on the transcriptome sequencing analysis, it was indicated that the upregulated phenylpropanoid biosynthesis-related genes in 84K poplar infected with C. gloeosporioides, including genes encoding PAL, C4H, 4CL, HCT, CCR, COMT, F5H, and CAD, are also involved in other KEGG pathways (i.e., flavonoid biosynthesis and phenylalanine metabolism). The expression levels of these genes were lowest in 84K-Cg and highest in 84K-DCg. CONCLUSIONS: It was found that PAL-related genes may be crucial for the induced resistance of 84K poplar to anthracnose, which enriched in the phenylpropanoid biosynthesis. These results will provide the basis for future research conducted to verify the contribution of phenylpropanoid biosynthesis to induced resistance and explore plant immune resistance-related signals that may regulate plant defense capabilities, which may provide valuable insights relevant to the development of effective and environmentally friendly methods for controlling poplar anthracnose.

Transcription factor CgSte12 regulates pathogenicity by affecting appressorium structural development in the anthracnose-causing fungus Colletotrichum gloeosporioides.

Colletotrichum gloeosporioides is the causal agent of poplar anthracnose, which induces major economic losses and adversely affects the ecosystem services of poplar forests. The appressorium serves as a penetration structure for many pathogenic fungi, including C. gloeosporioides. The production of mucilage and the formation of penetration pegs are critically important for the appressorium-mediated penetration of host tissues. We previously found that CgPmk1 is a key protein involved in appressorium formation, penetration, and pathogenicity. Although CgSte12, which is a transcription factor that functions downstream of CgPmk1, regulates the formation of penetration pegs, its role in C. gloeosporioides appressorium development and pathogenicity has not been elucidated. Here, we developed C. gloeosporioides CgSTE12 mutants and characterized the molecular and cellular functions of CgSTE12. The results showed that mycelial growth and morphology were not affected in the CgSTE12 knockout mutants, which produced normal melanized appressoria. However, these mutants had less mucilage secreted around the appressoria, impaired appressorial cone formation, and the inability to form penetration pores and pegs, which ultimately led to a significant loss of pathogenicity. Our comparative transcriptome analysis revealed that CgSte12 controls the expression of genes involved in appressorium development and function, including genes encoding cutinases, NADPH oxidase, spermine biosynthesis-related proteins, ceramide biosynthesis-related proteins, fatty acid metabolism-related proteins, and glycerophospholipid metabolism-related proteins. Overall, our findings indicate that CgSte12 is a critical regulator of appressorium development and affects C. gloeosporioides pathogenicity by modulating the structural integrity of appressoria.

Comparative Transcriptomic Analysis of MAPK-Mediated Regulation of Pathogenicity, Stress Responses, and Development in Cytospora chrysosperma.

Mitogen-activated protein kinase (MAPK) cascades are highly conserved signal transduction pathways that mediate cellular responses to various biotic and abiotic signals in plant-pathogenic fungi. Generally, there are three MAPKs in filamentous pathogenic fungi: Pmk1/Fus3/Kss1, Hog1, and Stl2. Our previous studies have shown that CcPmk1 is a core regulator of fungal pathogenicity in Cytospora chrysosperma, the causal agent of canker disease in a wide range of woody plants. Here, we identified and functionally characterized the other two MAPK genes (CcHog1 and CcSlt2) and then compared the transcriptional differences among these three MAPKs in C. chrysosperma. We found that the MAPKs shared convergent and distinct roles in fungal development, stress responses, and virulence. For example, CcHog1, CcSlt2, and CcPmk1 were all involved in conidiation and response to stresses, including hyperosmotic pressure, cell wall inhibition agents, and H2O2, but only CcPmk1 and CcSlt2 were required for hyphal growth and fungal pathogenicity. Transcriptomic analysis showed that numerous hyperosmosis- and cell wall-related genes significantly reduced their expression levels in ΔCcHog1 and ΔCcSlt2, respectively. Interestingly, RNA- and ribosome-related processes were significantly enriched in the upregulated genes of ΔCcSlt2, whereas they were significantly enriched in the downregulated genes of ΔCcPmk1. Moreover, two secondary metabolite gene clusters were significantly downregulated in ΔCcPmk1, ΔCcSlt2, and/or ΔCcHog1. Importantly, some virulence-associated genes were significantly downregulated in ΔCcPmk1 and/or ΔCcSlt2, such as candidate effector genes. Collectively, these results suggest that the similar and distinct phenotypes of each MAPK deletion mutant may result from the transcriptional regulation of a series of common or specific downstream genes, which provides a better understanding of the regulation network of MAPKs in C. chrysosperma.

Re-evaluation of the Fungal Diversity and Pathogenicity of Cytospora Species from Populus in China.

Poplar is widely cultivated in China because of its strong ecological adaptability, fast growth, easy reproduction, and short rotation period. However, it suffers from severe threat from canker disease caused by Cytospora species. The present study revealed the presence of Cytospora species from Populus in China. A total of six species of Cytospora were isolated from Populus in six provinces in China, including five known species (C. ailanthicola, C. chrysosperma, C. donglingensis, C. paratranslucens, and C. sophoriopsis) and one novel species (C. populi) based on morphological and phylogenetic analyses of ITS, act, rpb2, tef1-α, and tub2 gene sequences. Cytospora ailanthicola, C. chrysosperma, C. paratranslucens, and C. sophoriopsis are confirmed as pathogens by pathogenicity tests of which C. paratranslucens showed the strongest virulence, followed by C. ailanthicola, C. chrysosperma, and C. sophoriopsis. The mycelial growth rates of isolates from the six species had 22.5 to 27°C as the optimum temperatures, and the optimum pH values were 5.9 to 7.1. The effectiveness of six carbon sources on the mycelial growth showed that colonies grew the fastest in the presence of fructose and grew the slowest using xylose. This study represents a significant evaluation of Cytospora causing poplar canker disease in China.

Identification and Characterization of Neocosmospora silvicola Causing Canker Disease on Pinus armandii in China.

Chinese white pine, Pinus armandii, is a source of high-quality timber and an afforestation tree in China, which plays an important ecological and social role in water and soil conservation. Recently, a new canker disease has been reported in Longnan City, Gansu Province, where P. armandii is mainly distributed. In this study, the causal agent was isolated from diseased samples and identified as a fungal pathogen, Neocosmospora silvicola, based on morphological characteristics and molecular analyses (internal transcribed spacer, large subunit, RNA polymerase II, and translation elongation factor-1α). Pathogenicity tests on P. armandii revealed that N. silvicola isolates caused a 60% average mortality rate in artificially inoculated 2-year-old seedlings. The pathogenicity of these isolates was also observed on the branches of 10-year-old P. armandii trees with a 100% mortality rate. These results agree with the isolation of N. silvicola from diseased plants, suggesting the possible role of this fungus in the decline of P. armandii plants. Mycelial growth of N. silvicola was fastest on potato dextrose agar medium, and growth occurred at pH values ranging from 4.0 to 11.0 with temperatures between 5 and 40°C. The fungus also grew rapidly in complete darkness compared with other light conditions. Of the eight carbon and seven nitrogen sources tested, starch and sodium nitrate, respectively, were highly efficient in supporting the mycelial growth of N. silvicola. The ability of N. silvicola to grow at low temperatures (5°C) may explain its occurrence in the Longnan area of Gansu Province. This article is the first report of N. silvicola as an important fungal pathogen causing branch and stem cankers on Pinus tree species, which remains a threat to the forests.

Comparative Transcriptome Analysis Reveals the Effect of the DHN Melanin Biosynthesis Pathway on the Appressorium Turgor Pressure of the Poplar Anthracnose-Causing Fungus Colletotrichum gloeosporioides.

Anthracnose of poplar caused by Colletotrichum gloeosporioides is a leaf disease that seriously affects poplar growth. The pathogen invades the host in the form of adherent cells, which generate turgor pressure through the metabolism of intracellular substances prior to penetrating the epidermis of poplar leaves. In this study, the expansion-related pressure of the mature appressorium of the wild-type C. gloeosporioides was approximately 13.02 ± 1.54 MPa at 12 h, whereas it was 7.34 ± 1.23 MPa and 9.34 ± 2.22 MPa in the melanin synthesis-related gene knockout mutants ΔCgCmr1 and ΔCgPks1, respectively. The CgCmr1 and CgPks1 genes were highly expressed at 12 h in the wild-type control, implying that the DHN melanin biosynthesis pathway may play an important role in the mature appressorium stage. The transcriptome sequencing analysis indicated that the upregulated melanin biosynthesis genes in C. gloeosporioides, such as CgScd1, CgAyg1, CgThr1, CgThr2, and CgLac1, are involved in specific KEGG pathways (i.e., fatty acid biosynthesis, fatty acid metabolism, and biotin metabolism). Therefore, we speculate that the melanin synthesis-related genes and fatty acid metabolism pathway genes contribute to the regulation of the turgor pressure in the mature C. gloeosporioides appressorium, ultimately leading to the formation of infection pegs that enter plant tissues. These observations may reflect the co-evolution of C. gloeosporioides and its host.

Verification of the Protective Effects of Poplar Phenolic Compounds Against Poplar Anthracnose.

Poplar anthracnose caused by Colletotrichum gloeosporioides is one of the most important diseases widely distributed in poplar-growing areas in China, causing serious economic and ecological losses. In this study, three poplar species showed different resistance to poplar anthracnose: Populus × canadensis was resistant, Populus tomentosa was susceptible, and P. × beijingensis showed intermediate resistance. However, it remains uncertain whether phenolic compounds in poplar are involved in this resistance. Therefore, we determined the concentrations of phenolic compounds and their antifungal activity. Before and after the C. gloeosporioides inoculation, 20 phenolic compounds were detected in P. × canadensis and the number increased from 12 to 14 in P. × beijingensis but decreased from seven to four in P. tomentosa. Thus, phenolic compounds may be positively correlated with the degree of disease resistance. We selected seven phenolic compounds for further analysis, which varied considerably in content after inoculation with C. gloeosporioides. These seven compounds were salicin, arbutin, benzoic acid, salicylic acid, chlorogenic acid, ferulic acid, and naringenin, which helped poplar trees to limit the growth of C. gloeosporioides and differed in their antifungal effects, with phenolic acids having the strongest inhibitory effect. In addition, the optimal concentrations of different substances varied. We demonstrate that these phenolic compounds produced by poplar do play a certain role in the process of poplar resistance to anthracnose. These findings lay a foundation for future research into the antifungal mechanism of poplar trees and may be useful for enhancing the prevention and control of poplar anthracnose.

A Putative Effector CcSp84 of Cytospora chrysosperma Localizes to the Plant Nucleus to Trigger Plant Immunity.

Cytospora chrysosperma is the main causal agent of poplar canker disease in China, especially in some areas with poor site conditions. Pathogens secrete a large number of effectors to interfere the plant immunity and promote their infection and colonization. Nevertheless, the roles of effectors in C. chrysosperma remain poorly understood. In this study, we identified and functionally characterized a candidate effector CcSp84 from C. chrysosperma, which contained a nuclear localization signal motif at the C-terminal and was highly induced during infection stages. Transient expression of CcSp84 in Nicotiana benthamiana leaves could trigger cell death. Additionally, deletion of CcSp84 significantly reduced fungal virulence to the polar twigs, while no obvious defects were observed in fungal growth and sensitivity to H2O2. Confocal microscopy revealed that CcSp84 labeled with a green fluorescent protein (GFP) was mainly accumulated in the plant nucleus. Further analysis revealed that the plant nucleus localization of CcSp84 was necessary to trigger plant immune responses, including ROS accumulation, callose deposition, and induced expression of jasmonic acid and ethylene defense-related genes. Collectively, our results suggest that CcSp84 is a virulence-related effector, and plant nucleus localization is required for its functions.

High expression of LAMA3/AC245041.2 gene pair associated with KRAS mutation and poor survival in pancreatic adenocarcinoma: a comprehensive TCGA analysis.

BACKGROUND: Pancreatic adenocarcinoma (PAAD) is one of the most challenging cancers with high morbidity and mortality. KRAS mutations could occur as an early event in PAAD. The present study aimed to identify the differentially expressed lncRNAs (DE-lncRNAs) and differentially expressed mRNAs (DE-mRNAs) in KRAS-mutant PAAD to explore the pathogenesis and the underlying molecular mechanism of PAAD development. METHODS: Clinical data of TCGA-PAAD patients were downloaded from the TCGA database and subjected to survival analysis along with the KRAS mutation information data. Weighted gene correlation network analysis (WGCNA) and univariate Cox regression analysis were conducted to construct prognostic risk models to identify the hub DE-mRNAs and DE-lncRNAs associated with PAAD prognosis. GO and KEGG enrichment analyses of the identified hub DE-mRNAs were performed. Multivariate cox regression analysis was performed to analyze the overall prognosis of age, gender, pathologic_T, and KRAS mutations, following which the differences in the clinical characteristics of risk score1 and risk score2 were analyzed. Finally, the mRNAs-lncRNA-TFs regulatory network was constructed. RESULTS: Functional enrichment analysis was performed after screening 1671 DE-mRNAs and 324 DE-lncRNAs. It was observed that the associated pathways were enriched mainly in the modulation of chemical synaptic transmission, synaptic membrane, ion-gated channel activity, ligand-receptor interactions that stimulate neural tissue, among others. The univariate Cox regression analysis screened 117 mRNAs and 36 lncRNAs, and the risk ratio models of the mRNAs and lncRNAs were constructed. LAMA3 (mRNA) and AC245041.2 (lncRNA) exhibited a strong expression correlation in the respective two risk models. The genes in the samples with a high expression of these two genes were enriched in several pathways associated with transcription factors (TFs), among which the TFs ATF5, CSHL1, NR1I2, SIPA1, HOXC13, HSF2, and HOXA10 were shared by the two groups. The core enrichment genes in the common TF pathways were collated, and the mRNAs-lncRNAs-TFs regulatory network was constructed. CONCLUSION: In the present study, novel prognostic mRNAs and lncRNAs were identified, and their respective prognostic models and nomograms were constructed to guide clinical practice. An mRNAs-lncRNAs-TFs regulatory network was also constructed, which could assist further research in the future.

CgEnd3 Regulates Endocytosis, Appressorium Formation, and Virulence in the Poplar Anthracnose Fungus Colletotrichum gloeosporioides.

The hemibiotrophic ascomycete fungus Colletotrichum gloeosporioides is the causal agent of anthracnose on numerous plants, and it causes considerable economic losses worldwide. Endocytosis is an essential cellular process in eukaryotic cells, but its roles in C. gloeosporioides remain unknown. In our study, we identified an endocytosis-related protein, CgEnd3, and knocked it out via polyethylene glycol (PEG)-mediated protoplast transformation. The lack of CgEnd3 resulted in severe defects in endocytosis. C. gloeosporioides infects its host through a specialized structure called appressorium, and ΔCgEnd3 showed deficient appressorium formation, melanization, turgor pressure accumulation, penetration ability of appressorium, cellophane membrane penetration, and pathogenicity. CgEnd3 also affected oxidant adaptation and the expression of core effectors during the early stage of infection. CgEnd3 contains one EF hand domain and four calcium ion-binding sites, and it is involved in calcium signaling. A lack of CgEnd3 changed the responses to cell-wall integrity agents and fungicide fludioxonil. However, CgEnd3 regulated appressorium formation and endocytosis in a calcium signaling-independent manner. Taken together, these results demonstrate that CgEnd3 plays pleiotropic roles in endocytosis, calcium signaling, cell-wall integrity, appressorium formation, penetration, and pathogenicity in C. gloeosporioides, and it suggests that CgEnd3 or endocytosis-related genes function as promising antifungal targets.

CgHog1 controls the adaptation to both sorbitol and fludioxonil in Colletotrichum gloeosporioides.

The HOG (high-osmolarity glycerol) pathway is critical for the appropriate adaptation to adverse conditions. Here, we demonstrated that the deletion of CgHog1 resulted in enhanced sensitivity to osmotic stress and increased resistance to fludioxonil in the poplar anthracnose fungus Colletotrichum gloeosporioides. The accumulation of chitin around hyphal tips was obviously decreased in the ΔCgHog1 strain under sorbitol, whereas it strongly was increased in the response to fludioxonil compared with the wild type. To investigate the underlying mechanism of CgHog1-mediated adaption to osmotic stress and fludioxonil, transcriptomic profiles were performed in both the ΔCgHog1 strain and the wild type under the treatment of sorbitol and fludioxonil, respectively. Under sorbitol, genes associated with glycolysis, lipid metabolism, and accumulation of soluble sugars and amino acids were differentially expressed; under fludioxonil, vesicle trafficking-related genes were highly downregulated in the ΔCgHog1 strain, which was consistent with abnormal vacuoles distribution and morphology of hyphae, indicating that the growth defect caused by fludioxonil may be associated with disruption of endocytosis. Taken together, we elucidated the adaptation mechanisms of how CgHog1 regulates appropriate response to sorbitol and fludioxonil via different metabolism pathways. These findings extend our insights into the HOG pathway in fungi.

The bZIP transcription factor VdAtf1 regulates virulence by mediating nitrogen metabolism in Verticillium dahliae.

The fungus Verticillium dahliae causes vascular wilt disease on hundreds of plant species. Homologs of the bZIP transcription factor Atf1 are required for virulence in most pathogenic fungi, but the molecular basis for their involvement is largely unknown. We performed targeted gene deletion, expression analysis, biochemistry and pathogenicity assays to demonstrate that VdAtf1 governs pathogenesis via the regulation of nitrosative resistance and nitrogen metabolism in V. dahliae. VdAtf1 controls pathogenesis via the regulation of nitric oxide (NO) resistance and inorganic nitrogen metabolism rather than oxidative resistance and is important for penetration peg formation in V. dahliae. VdAtf1 affects ammonium and nitrate assimilation in response to various nitrogen sources. VdAtf1 may be involved in regulating the expression of VdNut1. VdAtf1 responds to NO stress by strengthening the fungal cell wall, and by causing over-accumulation of methylglyoxal and glycerol, which in turn impacts NO detoxification. We also verified that the VdAtf1 ortholog in Fusarium graminearum mediates nitrogen metabolism, suggesting conservation of this function in related plant pathogenic fungi. Our findings revealed new functions of VdAtf1 in pathogenesis, response to nitrosative stress and nitrogen metabolism in V. dahliae. The results provide novel insights into the regulatory mechanisms of the transcription factor VdAtf1 in virulence.

Genomewide Transcriptome Profiles Reveal How Bacillus subtilis Lipopeptides Inhibit Microsclerotia Formation in Verticillium dahliae.

Verticillium dahliae is a soilborne fungus and the primary causal agent of vascular wilt diseases worldwide. The fungus produces melanized microsclerotia that are crucially important for the survival and spread of V. dahliae. There are no fungicides available that are both effective and environmentally friendly to suppress the fungus. Previously, Bacillus subtilis C232 was isolated from soil and was demonstrated to suppress microsclerotia formation in V. dahliae. In this study, liquid chromatography coupled with mass spectrometry revealed that the antifungal substance is actually a mixture of lipopeptides. Exposure of V. dahliae to these lipopeptides resulted in hyphal swelling, cell lysis, and downregulation of melanin-related genes. RNA sequencing analyses of the lipopeptide-suppressed transcriptome during microsclerotial development revealed that 5,974 genes (2,131 upregulated and 3,843 downregulated) were differentially expressed versus nonsuppressive conditions. Furthermore, gene ontology enrichment analyses revealed that genes involved in response to stress, cellular metabolic processes, and translation were significantly enriched. Additionally, the lipopeptides inhibited expression of genes associated with secondary metabolism, protein catabolism, and the high-osmolarity glycerol response signaling pathway. Together, these findings provide evidence for the mechanism by which B. subtilis lipopeptides suppress microsclerotia formation. The transcriptomic insight garnered here may facilitate the development of biological agents to combat Verticillium wilt.

Insights into VdCmr1-mediated protection against high temperature stress and UV irradiation in Verticillium dahliae.

The fungus Verticillium dahliae causes vascular wilt disease on more than 200 plant species worldwide. This fungus can survive for years in soil as melanized microsclerotia. We found that VdCmr1, a transcription factor, is required for the melanin production and increased survival following UV irradiation in V. dahliae but not for microsclerotia production or virulence. Here, we provided evidence how VdCmr1 protects against high temperature (HT) and UV irradiation in V. dahliae. The results indicate that VdCmr1 mediates entry to the diapause period in V. dahliae in response to HT and contributes to the expression of proteins to minimize protein misfolding and denaturation. VdCmr1 deletion results in the misregulation of DNA repair machinery, suggestive of reduced DNA repair capacity following UV irradiation and in correlation with the low survival rate of UV-treated VdCmr1 mutants. We discovered a putative VdCmr1-dependent gene cluster associated with secondary metabolism and stress responses. We also functionally characterized two VdCmr1-responsive genes participating in HT and UV response. These results shed further light on the roles of VdCmr1 in protection from HT or UV irradiation, and the additional insights into the mechanisms of this protection may be useful to exploit for more effective disease control.

The mitogen-activated protein kinase gene CcPmk1 is required for fungal growth, cell wall integrity and pathogenicity in Cytospora chrysosperma.

Cytospora chrysosperma, the causal agent of canker disease in a wide range of woody plants, results in significant annual economic and ecological losses. Mitogen-activated protein kinase (MAPK) cascades are highly conserved signal transduction pathways that play a crucial role in mediating cellular responses to environmental and host signals in plant pathogenic fungi. In this study, we identified an ortholog of the Fus3/Kss1-related MAPK gene, CcPmk1, and characterized its functions in C. chrysosperma. The expression of CcPmk1 was highly induced by inoculation on poplar twigs, and targeted deletion of CcPmk1 resulted in the loss of pathogenicity, indicating that CcPmk1 is an important regulator of virulence. In addition, CcPmk1 deletion mutants (ΔCcPmk1) displayed reduced growth and conidiation, decreased fungal biomass production and hyperbranching. Furthermore, our results indicated that CcPmk1 deletion mutants exhibited hypersensitivity to cell wall inhibitors and cell wall-degrading enzymes. Correspondingly, the transcription of cell wall biosynthesis-related genes in the ΔCcPmk1 strain was downregulated compared to that in the wild-type strain. Moreover, we found that CcPmk1 could positively regulate the expression of several candidate effector encoding genes which were highly induced in planta. Hence, we hypothesized that CcPmk1 regulates the expression of a series of effectors to promote virulence. Overall, we concluded that the functions of CcPmk1 extend to fungal development, cell wall integrity and pathogenicity in C. chrysosperma.

A Cdc42 homolog in Colletotrichum gloeosporioides regulates morphological development and is required for ROS-mediated plant infection.

The Rho GTPase Cdc42 is conserved in fungi and plays a key role in regulating polarity establishment, morphogenesis and differentiation. In this study, we identified an ortholog of Cdc42, CgCdc42, and functionally characterized it to determine the role of Cdc42 in the development and pathogenicity of Colletotrichum gloeosporioides, a causal agent of poplar anthracnose. Targeted deletion of CgCdc42 resulted in reduced vegetative growth and dramatic morphological defects, including the formation of elongated conidia and abnormally shaped appressoria. Moreover, CgCdc42 deletion mutants were less virulent on poplar leaves than were wild type. Appressoria formed by ΔCgCdc42 mutants were morphologically abnormal and present in lower numbers on poplar leaves than were those formed by wild type. However, an ROS scavenging assay indicated that the ΔCgCdc42 mutants maintained wild type pathogenicity in the absence of ROS despite having fewer appressoria than wild type, suggesting that the ΔCgCdc42 mutants were deficient in their tolerance of ROS. Additionally, we also found that the distribution of ROS was different after the deletion of CgCdc42, the ΔCgCdc42 mutants were hypersensitive to H2O2, and transcriptional analysis revealed that CgCdc42 is involved in the regulation of ROS-related genes. Furthermore, loss of CgCdc42 caused defects in cell wall integrity and an uneven distribution of chitin. These data collectively suggest that CgCdc42 plays an important role in the regulation of vegetative growth, morphological development, cell wall integrity and ROS-mediated plant infection in C. gloeosporioides.

Development and Characterization of Novel Genic-SSR Markers in Apple-Juniper Rust Pathogen Gymnosporangium yamadae (Pucciniales: Pucciniaceae) Using Next-Generation Sequencing.

The Apple-Juniper rust, Gymnosporangium yamadae, is an economically important pathogen of apples and junipers in Asia. The absence of markers has hampered the study of the genetic diversity of this widespread pathogen. In our study, we developed twenty-two novel microsatellite markers for G. yamadae from randomly sequenced regions of the transcriptome, using next-generation sequencing methods. These polymorphic markers were also tested on 96 G. yamadae individuals from two geographical populations. The allele numbers ranged from 2 to 9 with an average value of 6 per locus. The polymorphism information content (PIC) values ranged from 0.099 to 0.782 with an average value of 0.48. Furthermore, the observed (HO) and expected (HE) heterozygosity ranged from 0.000 to 0.683 and 0.04 to 0.820, respectively. These novel developed microsatellites provide abundant molecular markers for investigating the genetic structure and genetic diversity of G. yamadae, which will help us to better understand disease epidemics and the origin and migration routes of the Apple-Juniper rust pathogen. Further studies will also be completed to dissect how human activities influence the formation of current population structures. Furthermore, these SSR (simple sequence repeat) markers can also be used as tools to identify virulence by mapping the whole genomes of different virulent populations. These markers will, thus, assist the development of effective risk-assessment models and management systems for the Apple-Juniper rust pathogen.

Comparative transcriptome analysis and identification of candidate effectors in two related rust species (Gymnosporangium yamadae and Gymnosporangium asiaticum).

BACKGROUND: Rust fungi constitute the largest group of plant fungal pathogens. However, a paucity of data, including genomic sequences, transcriptome sequences, and associated molecular markers, hinders the development of inhibitory compounds and prevents their analysis from an evolutionary perspective. Gymnosporangium yamadae and G. asiaticum are two closely related rust fungal species, which are ecologically and economically important pathogens that cause apple rust and pear rust, respectively, proved to be devastating to orchards. In this study, we investigated the transcriptomes of these two Gymnosporangium species during the telial stage of their lifecycles. The aim of this study was to understand the evolutionary patterns of these two related fungi and to identify genes that developed by selection. RESULTS: The transcriptomes of G. yamadae and G. asiaticum were generated from a mixture of RNA from three biological replicates of each species. We obtained 49,318 and 54,742 transcripts, with N50 values of 1957 and 1664, for G. yamadae and G. asiaticum, respectively. We also identified a repertoire of candidate effectors and other gene families associated with pathogenicity. A total of 4947 pairs of putative orthologues between the two species were identified. Estimation of the non-synonymous/synonymous substitution rate ratios for these orthologues identified 116 pairs with Ka/Ks values greater than1 that are under positive selection and 170 pairs with Ka/Ks values of 1 that are under neutral selection, whereas the remaining 4661 genes are subjected to purifying selection. We estimate that the divergence time between the two species is approximately 5.2 Mya. CONCLUSION: This study constitutes a de novo assembly and comparative analysis between the transcriptomes of the two rust species G. yamadae and G. asiaticum. The results identified several orthologous genes, and many expressed genes were identified by annotation. Our analysis of Ka/Ks ratios identified orthologous genes subjected to positive or purifying selection. An evolutionary analysis of these two species provided a relatively precise divergence time. Overall, the information obtained in this study increases the genetic resources available for research on the genetic diversity of the Gymnosporangium genus.

The two-component response regulator VdSkn7 plays key roles in microsclerotial development, stress resistance and virulence of Verticillium dahliae.

The fungus Verticillium dahliae causes vascular wilt disease on various plant species resulting in devastating yield losses worldwide. The capacity of V. dahliae to colonize in host plant xylem and disseminate by microsclerotia has led to studies to evaluate genes associated with pathogenesis and microsclerotia formation. Here, we identified and characterized a V. dahliae homolog to Skn7, a two-component stress response regulator of Saccharomyces cerevisiae. Results showed that melanized microsclerotia formation and conidiation were significantly inhibited in the VdSkn7 deletion mutants. VdSkn7-deficient mutants displayed severe growth defect under heat shock, cell wall perturbing agents and H2O2, and were significantly less virulent but were not sensitive to osmotic stresses compared to the wild-type strain. Finally, we demonstrated that VdSkn7 is required for the plant penetration. Taken together, our study thus provides new evidence on the functional conservation and divergence of Skn7 orthologs among fungal organisms and indicates that VdSkn7 contributes to microsclerotial development, virulence and stress response of V. dahliae.