The role of VdSti1 in Verticillium dahliae: insights into pathogenicity and stress responses.

Sti1/Hop, a stress-induced co-chaperone protein, serves as a crucial link between Hsp70 and Hsp90 during cellular stress responses. Despite its importance in stress defense mechanisms, the biological role of Sti1 in Verticillium dahliae, a destructive fungal pathogen, remains largely unexplored. This study focused on identifying and characterizing Sti1 homologues in V. dahliae by comparing them to those found in Saccharomyces cerevisiae. The results indicated that the VdSti1-deficient mutant displayed increased sensitivity to drugs targeting the ergosterol synthesis pathway, leading to a notable inhibition of ergosterol biosynthesis. Moreover, the mutant exhibited reduced production of microsclerotia and melanin, accompanied by decreased expression of microsclerotia and melanin-related genes VDH1, Vayg1, and VaflM. Additionally, the mutant's conidia showed more severe damage under heat shock conditions and displayed growth defects under various stressors such as temperature, SDS, and CR stress, as well as increased sensitivity to H2O2, while osmotic stress did not impact its growth. Importantly, the VdSti1-deficient mutant demonstrated significantly diminished pathogenicity compared to the wild-type strain. This study sheds light on the functional conservation and divergence of Sti1 homologues in fungal biology and underscores the critical role of VdSti1 in microsclerotia development, stress response, and pathogenicity of V. dahliae.

Correlation study on microbial communities and volatile flavor compounds in cigar tobacco leaves of diverse origins.

To elucidate the significant influence of microorganisms on geographically dependent flavor formation by analyzing microbial communities and volatile flavor compounds (VFCs) in cigar tobacco leaves (CTLs) obtained from China, Dominica, and Indonesia. Microbiome analysis revealed that the predominant bacteria in CTLs were Staphylococcus, Aerococcus, Pseudomonas, and Lactobacillus, while the predominant fungi were Aspergillus, Wallemia, and Sampaiozyma. The microbial communities of CTLs from different origins differed to some extent, and the diversity and abundance of bacteria were greater than fungi. Metabolomic analysis revealed that 64 VFCs were identified, mainly ketones, of which 23 VFCs could be utilized to identify the geographical origins of CTLs. Sixteen VFCs with OAV greater than 1, including cedrol, phenylacetaldehyde, damascone, beta-damascone, and beta-ionone, play important roles in shaping the flavor profile of CTLs from different origins. Combined with the correlation analysis, bacterial microorganisms were more closely related to key VFCs and favored a positive correlation. Bacillus, Vibrio, and Sphingomonas were the main flavor-related bacteria. The study demonstrated that the predominant microorganisms were essential for the formation of key flavor qualities in CTLs, which provided a theoretical reference for flavor control of CTLs by microbial technology. KEY POINTS: • It is the high OAV VFCs that determine the flavor profile of CTLs. • The methylerythritol phosphate (MEP) pathway and the carotenoid synthesis pathway are key metabolic pathways for the formation of VFCs in CTLs. • Microbial interactions influence tobacco flavor, with bacterial microorganisms contributing more to the flavor formation of CTLs.

Temperature impacts on cotton yield superposed by effects on plant growth and verticillium wilt infection in China.

China produces and consumes the largest amount of cotton, playing a critical role in the world's fiber and textile industries. Theoretically, an increase in temperature poses a complex set of impacts on both cotton and pathogen diseases. However, empirical evidence regarding the overall effect on regional cotton yield in China is currently lacking. In this study, we employ county-level cotton statistics and degree-day indices (n = 30,502) to demonstrate a temperature effect on cotton yield, influenced by both direct temperature effects and indirect effects on verticillium wilt infection in China. Our findings indicate that temperatures between the base growing temperature (15 °C) and the optimal infection threshold for cotton wilt disease (25 °C) reduce cotton yield. However, beyond this threshold, when disease infection is significantly limited, higher temperatures become beneficial. Temperatures exceeding 32 °C causes heat stress, which dominates and drives a decline in yield. Furthermore, we provide a risk assessment of warming on cotton in future climate scenarios. Our model projections reveal an overall decrease in cotton yield ranging from 6.2 to 30.6%, accompanied by amplified heat stress (resulting in a yield decrease of 11.6 to 48.7%) but a reduced threat of verticillium wilt (yield increase of 8.2 to 23.6%) in future. Particularly, the Northwest Region, currently responsible for 80% of cotton production, is expected to be particularly vulnerable. This study emphasizes the importance of investing in long-term technological advancements such as cotton heat-tolerance breeding and redistributing cotton growing areas.

Integrated microbiology and metabolomics analysis reveal responses of cotton rhizosphere microbiome and metabolite spectrum to conventional seed coating agents.

Fludioxonil (FL) and metalaxyl-M·fludioxonil·azoxystrobin (MFA) are conventional seed coating agents for controlling cotton seedling diseases. However, their effects on seed endophytic and rhizosphere microecology are still poorly understood. This study aimed to assess the effects of FL and MFA on cotton seed endophytes, rhizosphere soil enzymatic activities, microbiome and metabolites. Both seed coating agents significantly changed seed endophytic bacterial and fungal communities. Growing coated seeds in the soils originating from the Alar (AL) and Shihezi (SH) region inhibited soil catalase activity and decreased both bacterial and fungal biomass. Seed coating agents increased rhizosphere bacterial alpha diversity for the first 21 days but decreased fungal alpha diversity after day 21 in the AL soil. Seed coating reduced the abundance of a number of beneficial microorganisms but enriched some potential pollutant-degrading microorganisms. Seed coating agents may have affected the complexity of the co-occurrence network of the microbiome in the AL soil, reducing connectivity, opposite to what was observed in the SH soil. MFA had more pronounced effects on soil metabolic activities than FL. Furthermore, there were strong links between soil microbial communities, metabolites and enzymatic activities. These findings provide valuable information for future research and development on application of seed coatings for disease management.

Acetylation of GhCaM7 enhances cotton resistance to Verticillium dahliae.

Protein lysine acetylation is an important post-translational modification mechanism involved in cellular regulation in eukaryotes. Calmodulin (CaM) is a ubiquitous Ca2+ sensor in eukaryotes and is crucial for plant immunity, but it is so far unclear whether acetylation is involved in CaM-mediated plant immunity. Here, we found that GhCaM7 is acetylated upon Verticillium dahliae (V. dahliae) infection and a positive regulator of V. dahliae resistance. Overexpressing GhCaM7 in cotton and Arabidopsis enhances V. dahliae resistance and knocking-down GhCaM7 makes cotton more susceptible to V. dahliae. Transgenic Arabidopsis plants overexpressing GhCaM7 with mutation at the acetylation site are more susceptible to V. dahliae than transgenics overexpressing the wild-type GhCaM7, implying the importance of the acetylated GhCaM7 in response to V. dahliae infection. Yeast two-hybrid, bimolecular fluorescent complementation, luciferase complementation imaging, and coimmunoprecipitation assays demonstrated interaction between GhCaM7 and an osmotin protein GhOSM34 that was shown to have a positive role in V. dahliae resistance. GhCaM7 and GhOSM34 are co-localized in the cell membrane. Upon V. dahliae infection, the Ca2+ content reduces almost instantly in plants with downregulated GhCaM7 or GhOSM34. Down regulating GhOSM34 enhances accumulation of Na+ and increases cell osmotic pressure. Comparative transcriptomic analyses between cotton plants with an increased or reduced expression level of GhCaM7 and wild-type plants indicate the involvement of jasmonic acid signaling pathways and reactive oxygen species in GhCaM7-enabled disease resistance. Together, these results demonstrate the involvement of CaM protein in the interaction between cotton and V. dahliae, and more importantly, the involvement of the acetylated CaM in the interaction.

Soil bacterial community response to continuous cropping of cotton.

Introduction: Long-term continuous cropping may result in the outbreak and proliferation of soil-borne diseases, as well as reduction in annual crop production. Overcoming the obstacles of continuous cropping is critical for the long-term growth of modern agriculture. Soil microbes are essential for plant health, but the consequences of continuous cropping on soil microbiome are still poorly understood. Methods: This study analyzed changes in soil bacterial community composition of Aksu (AKS) and Shihezi (SHZ) in Xinjiang Province during 1-20 years of continuous cropping by 16S amplicon sequencing. The results showed that the incidence of cotton Verticillium wilt rose with the number of cropping years. The bacterial alpha diversity in the AKS soil grew as the number of continuous cropping years increased, however it declined in the SHZ soil. Results: The results of beta diversity analysis showed that there were significant differences in soil bacterial communities between different continuous cropping years and between different soils. The results of community composition changes at the level of main phyla and genus showed that the relative abundance of Actinobacteria, Bacteroidetes and Streptomyces decreased with the increase of continuous cropping years in the AKS and the SHZ soils. In addition, Actinobacteria, Propionibacteriales, and Nocardioidaceae were significantly enriched during the early stages of continuous cropping. Network analysis showed that long-term (≥8 years) continuous cropping interfered with the complexity of soil bacterial co-occurrence networks and reduced collaboration between OTUs. Discussion: These findings suggested that continuous cropping and soil origin jointly affected the diversity and structural of bacterial communities, and the loss of Nocardioidaceae and Streptomyces in Actinobacteria might be one of the reasons of continuous cropping obstacles.

Galactosyltransferase GhRFS6 interacting with GhOPR9 involved in defense against Verticillium wilt in cotton.

The soil-borne fungus Verticillium dahliae causes Verticillium wilt (VW), one of the most devastating diseases of cotton. In a previous study showed that GhOPR9 played a positive role in resistance of cotton to VW through the regulation of the Jasmonic acid (JA) pathway. Furtherly, we also found that GhOPR9 interacted with a sucrose galactosyltransferase GhRFS6. Raffinose synthase (RFS) plays a key role in plant innate immunity, including the abiotic stress of drought, darkness. However, there were few reports on the effects of RFS on biotic stress. In this study, we verified the function of GhRFS6 to VW. The expression analysis showed that the GhRFS6 may be regulated by various stresses, and it was upregulated under Vd076 and Vd991 pressures. Inhibition of GhRFS6 expression, hydrogen peroxide (H2O2) content, lignin content, cell wall thickness and a series of defense responses were decreased, and the resistance of cotton to V. dahliae was decreased. In addition, this study showed that GhRFS6 has glycosyltransferase activity and can participate in the regulation of α-galactosidase activity and raffinose and inositol synthesis. And that galactose was accumulated in cotton roots after GhRFS6 silencing, which is beneficial for the colonization and growth of V. dahliae. Furthermore, overexpression of GhRFS6 in Arabidopsis thaliana enhanced plant resistance to V. dahliae. In GUS staining, the promoter expression position of GhRFS6 was also altered after V. dahliae infection. Meanwhile, GhRFS6 has also been shown to resist VW through the regulation of the JA pathway. These results suggest that GhRFS6 is a potential molecular target for improving cotton resistance to VW.

Transcriptome Analysis Reveals the Defense Mechanism of Cotton against Verticillium dahliae Induced by Hypovirulent Fungus Gibellulopsis nigrescens CEF08111.

Verticillium wilt is a kind of plant vascular disease caused by the soilborne fungus Verticillium dahliae, which severely limits cotton production. Our previous studies showed that the endophytic fungus Gibellulopsis nigrescens CEF08111 can effectively control Verticillium wilt and induce a defense response in cotton plants. However, the comprehensive molecular mechanism governing this response is not yet clear. To study the signaling mechanism induced by strain CEF08111, the transcriptome of cotton seedlings pretreated with CEF08111 was sequenced. The results revealed 249, 3559 and 33 differentially expressed genes (DEGs) at 3, 12 and 48 h post inoculation with CEF08111, respectively. At 12 h post inoculation with CEF08111, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that the DEGs were enriched mainly in the plant−pathogen interaction, mitogen-activated protein kinase (MAPK) signaling pathway-plant, and plant hormone signal transduction pathways. Gene ontology (GO) analysis revealed that these DEGs were enriched mainly in the following terms: response to external stimulus, systemic acquired resistance, kinase activity, phosphotransferase activity, xyloglucan: xyloglucosyl transferase activity, xyloglucan metabolic process, cell wall polysaccharide metabolic process and hemicellulose metabolic process. Moreover, many genes, such as calcium-dependent protein kinase (CDPK), flagellin-sensing 2 (FLS2), resistance to Pseudomonas syringae pv. maculicola 1(RPM1) and myelocytomatosis protein 2 (MYC2), that regulate crucial points in defense-related pathways were identified and may contribute to V. dahliae resistance in cotton. Seven DEGs of the pathway phenylpropanoid biosynthesis were identified by weighted gene co-expression network analysis (WGCNA), and these genes are related to lignin synthesis. The above genes were compared and analyzed, a total of 710 candidate genes that may be related to the resistance of cotton to Verticillium wilt were identified. These results provide a basis for understanding the molecular mechanism by which the biocontrol fungus CEF08111 increases the resistance of cotton to Verticillium wilt.

VdERG2 was involved in ergosterol biosynthesis, nutritional differentiation and virulence of Verticillium dahliae.

The ergosterol biosynthesis pathway plays an important role in model pathogenic bacteria Saccharomyces cerevisiae, but little is known about the biosynthesis of ergosterol in the pathogenic fungus Verticillium dahliae. In this study, we identified the VdERG2 gene encoding sterol C-8 isomerase from V. dahliae and investigated its function in virulence by generating gene deletion mutants (ΔVdERG2) and complemented mutants (C-ΔVdERG2). Knockout of VdERG2 reduced ergosterol content. The conidial germination rate and conidial yield of ΔVdERG2 significantly decreased and abnormal conidia were produced. In spite of VdERG2 did not affect the utilization of carbon sources by V. dahliae, but the melanin production of ΔVdERG2 was decreased in cellulose and pectin were used as the sole carbon sources. Furthermore, the ΔVdERG2 mutants produced less microsclerotia and melanin with a significant decrease in the expression of microsclerotia and melanin-related genes VaflM, Vayg1, VDH1, VdLAC, VdSCD and VT4HR. In addition, mutants ΔVdERG2 were very sensitive to congo red (CR), sodium dodecyl sulfate (SDS) and hydrogen peroxide (H2O2) stresses, indicating that VdERG2 was involved in the cell wall and oxidative stress response. The absence of VdERG2 weakened the penetration ability of mycelium on cellophane and affected the growth of mycelium. Although ΔVdERG2 could infect cotton, its pathogenicity was significantly impaired. These phenotypic defects in ΔVdERG2 could be complemented by the reintroduction of a full-length VdERG2 gene. In summary, as a single conservative secretory protein, VdERG2 played a crucial role in ergosterol biosynthesis, nutritional differentiation and virulence in V. dahliae.

VdGAL4 Modulates Microsclerotium Formation, Conidial Morphology, and Germination To Promote Virulence in Verticillium dahliae.

Verticillium dahliae Kleb is a typical soilborne pathogen that can cause vascular wilt disease on more than 400 plants. Functional analysis of genes related to the growth and virulence is crucial to revealing the molecular mechanism of the pathogenicity of V. dahliae. Glycosidase hydrolases can hydrolyze the glycosidic bond, and some can cause host plant immune response to V. dahliae. Here, we reported a functional validation of VdGAL4 as an α-galactosidase that belongs to glycoside hydrolase family 27. VdGAL4 could cause plant cell death, and its signal peptide plays an important role in cellular immune response. VdGAL4-triggered cell death depends on BAK1 and SOBIR1 in Nicotiana benthamiana. In V. dahliae, the function of VdGAL4 in mycelial growth, conidia, microsclerotium, and pathogenicity was studied by constructing VdGAL4 deletion and complementation mutants. Results showed that the deletion of VdGAL4 reduced the conidial yield and conidial germination rate of V. dahliae and changed the microscopic morphology of conidia; the mycelia were arranged more disorderly and were unable to produce microsclerotium. The VdGAL4 deletion mutants exhibited reduced utilization of different carbon sources, such as raffinose and sucrose. The VdGAL4 deletion mutants were also more sensitive to abiotic stress agents of SDS, sorbitol, low-temperature stress of 16°C, and high-temperature stress of 45°C. In addition, the VdGAL4 deletion mutants lost the ability to penetrate cellophane and its mycelium were disorderly arranged. Remarkably, VdGAL4 deletion mutants exhibited reduced pathogenicity of V. dahliae. These results showed that VdGAL4 played a critical role in the pathogenicity of V. dahliae by regulating mycelial growth, conidial morphology, and the formation of microsclerotium. IMPORTANCE This study showed that α-galactosidase VdGAL4 of V. dahliae could activate plant immune response and plays an important role in conidial morphology and yield, formation of microsclerotia, and mycelial penetration. VdGAL4 deletion mutants significantly reduced the pathogenicity of V. dahliae. These findings deepened the understanding of pathogenic virulence factors and how the mechanism of pathogenic fungi infected the host, which may help to seek new strategies for effective control of plant diseases caused by pathogenic fungi.

Two Metalloproteases VdM35-1 and VdASPF2 from Verticillium dahliae Are Required for Fungal Pathogenicity, Stress Adaptation, and Activating Immune Response of Host.

Verticillium dahliae is a soilborne fungus that causes destructive vascular wilt diseases in a wide range of plant hosts. In this study, we identified two M35 family metalloproteinases: VdM35-1 and VdASPF2, and investigated their function in vitro and in vivo. The results showed that VdM35-1 and VdASPF2 were located in the cell membrane, as secreted proteins depended on signal peptide, and two histidine residues (H) induced cell death and activated plant immune response. VdM35-1 depended on membrane receptor proteins NbBAK1 and NbSOBIR1 in the process of inducing cell death, while VdASPF2 did not depend on them. The deletion of VdM35-1 and VdASPF2 led to the decrease of sporulation and the slow shortening of mycelial branch growth, and the spore morphology of VdM35-1-deficient strain became malformed. In addition, ΔVdM35-1 and ΔVdASPF2 showed more sensitive to osmotic stress, SDS, Congo red (CR), and high temperature. In terms of the utilization of carbon sources, the knockout mutants exhibited decreased utilization of carbon sources, and the growth rates on the medium containing sucrose, starch, and pectin were lower than the wild type strain, with significantly limited growth, especially on galactose-containing medium. Furthermore, ΔVdM35-1 and ΔVdASPF2 showed a significant reduction in pathogenicity. Collectively, these results suggested that VdM35-1 and VdASPF2 were important multifunction factors in the pathogenicity of V. dahliae and relative to stress adaptation and activated plant immune response. IMPORTANCE Verticillium wilt, caused by the notorious fungal pathogen V. dahliae, is one of the main limiting factors for agricultural production. Metalloproteases played an important role in the pathogenic mechanism of pathogens. Our research found that M35 family metalloproteases VdM35-1 and VdASPF2 played an important role in the development, adaptability, and pathogenicity of V. dahliae, providing a new perspective for further understanding the molecular mechanism of virulence of fungal pathogens.

Identification and Functional Analysis of a Novel Hydrophobic Protein VdHP1 from Verticillium dahliae.

Verticillium dahliae could cause destructive vascular wilt disease on hundreds of plant species around the world, including cotton. In this study, we characterized the function of a hydrophobin gene VdHP1 in pathogen development and pathogenicity. Results showed that VdHP1 could induce cell death and activate plant immune responses. The VdHP1 deletion mutants (ΔVdHP1) and the complement mutants (C-ΔVdHP1) were obtained by the homologous recombination method. The VdHP1 deletion mutants exhibited increased hydrophilicity, inhibited microsclerotial formation, and reduced spore smoothness. In addition, the deletion mutants were more sensitive to NaCl, while relatively insensitive to KCl and sorbitol. Mutants also had greater resistance to Congo red, UV radiation, and high temperature, which suggested that ΔVdHP1 strains have stronger resistance to abiotic stress in general. Different carbon source assays showed that the utilization ability of skim milk, cellulose, and starch was greatly enhanced in ΔVdHP1, compared with that of WT and complemented strains. Furthermore, VdHP1 did not affect mycelium penetration on cellophane but contributed to mycelium growth on surface of the living plant cells. The pathogenicity test found that the crude toxin content, colonization, and dispersal of ΔVdHP1 was significantly increased compared with the WT and complementary strains. In addition, cotton seedlings showed more severe wilting symptoms after inoculation with ΔVdHP1 strains. These results suggested that the hydrophobin VdHP1 negatively regulated the virulence of V. dahliae, and played an important role in development, adaptability, and pathogenicity in V. dahliae, which maybe provide a new viewpoint to further understand the molecular mechanisms of pathogen virulence. IMPORTANCE Verticillium dahliae is a soilborne fungal pathogen that causes a destructive vascular disease on a large number of plant hosts, resulting in great threat to agricultural production. In this study, it was illustrated that the hydrophobin VdHP1 could induce cell death and activate plant immune responses. VdHP1 affected the hydrophobicity of V. dahliae, and negatively regulated the strains resistant to stress, and the utilization ability of different carbon sources. In addition, VdHP1 did not affect mycelium penetration on cellophane but contributed to mycelium growth on surface of the living plant cells. The VdHP1 gene negatively regulated the total virulence, colonization, and dispersal of V. dahliae, with enhanced pathogenicity of mutant strains in this gene. These results suggested that the hydrophobin VdHP1 played an importance in development, adaptability, and pathogenicity in V. dahliae, and would provide a new viewpoint to further understand the molecular mechanisms of pathogen virulence.

A DEK domain-containing protein GhDEK2D mediated Gossypium hirsutum enhanced resistance to Verticillium dahliae.

DEK is associated with DNA replication and break repair, mRNA splicing, and transcriptional regulation, which had been studied in humans and mammals. The function of DEK in plants was poorly understood. In this study, GhDEK2D was identified in Gossypium hirsutum by genome-wide and post-translational modifications. GhDEK2D had been phosphorylated, acetylated and ubiquitylated under Verticillium dahliae (Vd) challenge. The GhDEK2D-silenced cotton decreased resistance against Vd. In GhDEK2D-silenced cotton plants, the reactive oxygen species was activated, the callose, xylogen, hypersensitive reaction (HR) and expression levels of defense-related genes were reduced. Homozygous overexpressing-GhDEK2D transgenic Arabidopsis lines were more resistant to Verticillium wilt (Vw). We propose that GhDEK2D was a potential molecular target for improving resistance to Vw in cotton.

MPK homolog GhNTF6 was involved in cotton against Verticillium wilt by interacted with VdEPG1.

Mitogen-activated protein kinases (MPKs) are important in regulating plant development and stress response. Rapid activation of MPKs in plants usually depends on its phosphorylated. In view of this situation, a phosphorylated GhNTF6 belonged to MPKs family was screened in cotton roots under Verticillium dahliae challenge by phosphoproteomics analysis. Expression of GhNTF6 in cotton plants was did not induce by V. dahliae infection, while, silencing GhNTF6 results to enhance cotton plants susceptibility to V. dahliae, overexpression - GhNTF6 enhance Arabidopsis plants survivability to V. dahliae. Moreover, the mutation of GhNTF6 at site Thr195 and Thy197 with the phosphorylation decreased the plant resistance to V. dahliae. Therefore, GhNTF6 phosphorylation is important in plants against V. dahliae. Further analysis demonstrated that GhNTF6 interacted with a V. dahliae endopolygalacturonase (VdEPG1) on the cell nucleus. We propose that GhNTF6 is a potential molecular target for improving resistance to Verticillium wilt in cotton.

The Respiratory Burst Oxidase Homolog Protein D (GhRbohD) Positively Regulates the Cotton Resistance to Verticillium dahliae.

Verticillium wilt, mainly caused by a soil-inhabiting fungus Verticillium dahliae, can seriously reduce the yield and quality of cotton. The complex mechanism underlying cotton resistance to Verticillium wilt remains largely unknown. In plants, reactive oxygen species (ROS) mediated by Rbohs is one of the earliest responses of plants to biotic and abiotic stresses. In our previous study, we performed a time-course phospho-proteomic analysis of roots of resistant and susceptible cotton varieties in response to V. dahliae, and found early differentially expressed protein burst oxidase homolog protein D (GhRbohD). However, the role of GhRbohD-mediated ROS in cotton defense against V. dahliae needs further investigation. In this study, we analyzed the function of GhRbohD-mediated resistance of cotton against V. dahliae in vitro and in vivo. Bioinformatics analysis showed that GhRbohD possessed the conservative structural attributes of Rbohs family, 12 members of RbohD out of 57 Rbohs in cotton. The expression of GhRbohD was significantly upregulated after V. dahliae inoculation, peaking at 6 hpi, and the phosphorylation level was also increased. A VIGS test demonstrated that ROS production, NO, H2O2 and Ca2+ contents of GhRbohD-silenced cotton plants were significantly reduced, and lignin synthesis and callose accumulation were damaged, important reasons for the impairment of GhRbohD-silenced cotton's defense against V. dahliae. The expression levels of resistance-related genes were downregulated in GhRbohD-silenced cotton by qRT-PCR, mainly involving the lignin metabolism pathway and the jasmonic acid signaling pathway. However, overexpression of GhRbohD enhanced resistance of transgenic Arabidopsis to V. dahliae challenge. Furthermore, Y2H assays were applied to find that GhPBL9 and GhRPL12C may interact with GhRbohD. These results strongly support that GhRbohD activates ROS production to positively regulate the resistance of plants against V. dahliae.

Genome-Wide Analysis of Ribosomal Protein GhRPS6 and Its Role in Cotton Verticillium Wilt Resistance.

Verticillium wilt is threatening the world's cotton production. The pathogenic fungus Verticillium dahliae can survive in the soil in the form of microsclerotia for a long time, colonize through the root of cotton, and invade into vascular bundles, causing yellowing and wilting of cotton leaves, and in serious cases, leading to plant death. Breeding resistant varieties is the most economical and effective method to control Verticillium wilt. In previous studies, proteomic analysis was carried out on different cotton varieties inoculated with V. dahliae strain Vd080. It was found that GhRPS6 was phosphorylated after inoculation, and the phosphorylation level in resistant cultivars was 1.5 times than that in susceptible cultivars. In this study, knockdown of GhRPS6 expression results in the reduction of SA and JA content, and suppresses a series of defensive response, enhancing cotton plants susceptibility to V. dahliae. Overexpression in Arabidopsis thaliana transgenic plants was found to be more resistant to V. dahliae. Further, serines at 237 and 240 were mutated to phenylalanine, respectively and jointly. The transgenic Arabidopsis plants demonstrated that seri-237 compromised the plant resistance to V. dahliae. Subcellular localization in Nicotiana benthamiana showed that GhRPS6 was localized in the nucleus. Additionally, the pathogen inoculation and phosphorylation site mutation did not change its localization. These results indicate that GhRPS6 is a potential molecular target for improving resistance to Verticillium wilt in cotton. This lays a foundation for breeding disease-resistant varieties.

Calcium-Dependent Protein Kinase GhCDPK28 Was Dentified and Involved in Verticillium Wilt Resistance in Cotton.

Verticillium dahliae is a soil-borne fungus that causes vascular wilt through the roots of plants. Verticillium wilt caused by V. dahliae is one of the main diseases in cotton producing areas of the world, resulting in huge economic losses. Breeding resistant varieties is the most economical and effective method to control Verticillium wilt. Calcium-dependent protein kinases (CDPKs) play a pivotal role in plant innate immunity, including regulation of oxidative burst, gene expression as well as hormone signal transduction. However, the function of cotton CDPKs in response to V. dahliae stress remains unexplored. In this study, 96, 44 and 57 CDPKs were identified from Gossypium hirsutum, Gossypium raimondii and Gossypium arboretum, respectively. Phylogenetic analysis showed that these CDPKs could be divided into four branches. All GhCDPKs of the same clade are generally similar in gene structure and conserved domain arrangement. Cis-acting elements related to hormones, stress response, cell cycle and development were predicted in the promoter region. The expression of GhCDPKs could be regulated by various stresses. Gh_D11G188500.1 and Gh_A11G186100.1 was up-regulated under Vd0738 and Vd991 stress. Further phosphoproteomics analysis showed that Gh_A11G186100.1 (named as GhCDPK28-6) was phosphorylated under the stress of V. dahliae. Knockdown of GhCDPK28-6 expression, the content of reactive oxygen species was increased, a series of defense responses were enhanced, and the sensitivity of cotton to V. dahliae was reduced. Moreover, overexpression of GhCDPK28-6 in Arabidopsis thaliana weakened the resistance of plants to this pathogen. Subcellular localization revealed that GhCDPK28-6 was localized in the cell membrane. We also found that GhPBL9 and GhRPL12C may interact with GhCDPK28-6. These results indicate that GhCDPK28-6 is a potential molecular target for improving resistance to Verticillium wilt in cotton. This lays a foundation for breeding disease-resistant varieties.

A cotton WAKL protein interacted with a DnaJ protein and was involved in defense against Verticillium dahliae.

Accumulating evidence indicates that plant cell wall-associated receptor-like kinases (WAKs) involve in defense against pathogen attack, but their related signaling processes and regulatory mechanism remain largely unknown. We identified a WAK-like kinase (GhWAKL) from upland cotton (Gossypium hirsutum) and characterized its functional mechanism. Expression of GhWAKL in cotton plants was induced by Verticillium dahliae infection and responded to the application of salicylic acid (SA). Knockdown of GhWAKL expression results in the reduction of SA content and suppresses the SA-mediated defense response, enhancing cotton plants susceptibility to V. dahliae. And, ecotopic overexpression of GhWAKL in Arabidopsis thaliana conferred plant resistance to the pathogen. Further analysis demonstrated that GhWAKL interacted with a cotton DnaJ protein (GhDNAJ1) on the cell membrane. Silencing GhDNAJ1 also enhanced cotton susceptibility to V. dahliae. Moreover, the mutation of GhWAKL at site Ser628 with the phosphorylation decreased the interaction with GhDNAJ1 and compromised the plant resistance to V. dahliae. We propose that GhWAKL is a potential molecular target for improving resistance to Verticillium wilt in cotton.

CGTase, a novel antimicrobial protein from Bacillus cereus YUPP-10, suppresses Verticillium dahliae and mediates plant defence responses.

Verticillium wilt is a plant vascular disease caused by the soilborne fungus Verticillium dahliae that severely limits cotton production. In a previous study, we screened Bacillus cereus YUPP-10, an efficient antagonistic bacterium, to uncover mechanisms for controlling verticillium wilt. Here, we report a novel antimicrobial cyclodextrin glycosyltransferase (CGTase) from YUPP-10. Compared to other CGTases, six different conserved domains were identified, and six mutants were constructed by gene splicing with overlap extension PCR. Functional analysis showed that domain D was important for hydrolysis activity and domains A1 and C were important for inducing disease resistance. Direct effects of recombinant CGTase on V. dahliae included reduced mycelial growth, spore germination, spore production, and microsclerotia germination. In addition, CGTase also elicited cotton's innate defence reactions. Transgenic Arabidopsis thaliana lines that overexpress CGTase showed higher resistance to verticillium wilt. Transgenic CGTase A. thaliana plants grew faster and resisted disease better. CGTase overexpression enabled a burst of reactive oxygen species production and activated pathogenesis-related gene expression, indicating that the transgenic cotton was better prepared to protect itself from infection. Our work revealed that CGTase could inhibit the growth of V. dahliae, activate innate immunity, and play a major role in the biocontrol of fungal pathogens.

Fusarium oxysporum KB-3 from Bletilla striata: an orchid mycorrhizal fungus.

Orchid mycorrhizal fungi are essential for the seed germination and vegetative growth of orchids. The orchid Bletilla striata has great medical value in China because its tuber is rich in mannan. Some endophytic fungi were isolated from the roots of B. striata. The isolate KB-3 was selected for experiments because it could promote the germination of B. striata seeds. Based on morphological characters and phylogenetic analysis, the isolate KB-3 was identified as Fusarium oxysporum. Co-cultivation experiments of KB-3 with B. striata and Dendrobium candidum were performed to demonstrate orchid mycorrhizal structures. Microscopic examination showed that KB-3 established colonization and produced coiled hyphal structures known as pelotons within the cortical cells of both orchid roots. The results confirm that F. oxysporum KB-3 can behave as an orchid mycorrhizal fungus.