Analysis of endophytic bacterial diversity in seeds of different genotypes of cotton and the suppression of Verticillium wilt pathogen infection by a synthetic microbial community.

BACKGROUND: In agricultural production, fungal diseases significantly impact the yield and quality of cotton (Gossypium spp.) with Verticillium wilt posing a particularly severe threat. RESULTS: This study is focused on investigating the effectiveness of endophytic microbial communities present in the seeds of disease-resistant cotton genotypes in the control of cotton Verticillium wilt. The technique of 16S ribosomal RNA (16S rRNA) amplicon sequencing identified a significant enrichment of the Bacillus genus in the resistant genotype Xinluzao 78, which differed from the endophytic bacterial community structure in the susceptible genotype Xinluzao 63. Specific enriched strains were isolated and screened from the seeds of Xinluzao 78 to further explore the biological functions of seed endophytes. A synthetic microbial community (SynCom) was constructed using the broken-rod model, and seeds of the susceptible genotype Xinluzao 63 in this community that had been soaked with the SynCom were found to significantly control the occurrence of Verticillium wilt and regulate the growth of cotton plants. Antibiotic screening techniques were used to preliminarily identify the colonization of strains in the community. These techniques revealed that the strains can colonize plant tissues and occupy ecological niches in cotton tissues through a priority effect, which prevents infection by pathogens. CONCLUSION: This study highlights the key role of seed endophytes in driving plant disease defense and provides a theoretical basis for the future application of SynComs in agriculture.

Development and validation of allele-specific PCR-based SNP typing in a gene on chromosome D03 conferring resistance to Fusarium wilt race 4 in Upland cotton (Gossypium hirsutum).

Upland cotton (Gossypium hirsutum) is the most important fiber crop for the global textile industry. Fusarium oxysporum f. sp. vasinfectum (FOV) is one of the most destructive soil-borne fungal pathogens in cotton. Among eight pathogenic races and other strains, FOV race 4 (FOV4) is the most virulent race in US cotton production. A single nucleotide polymorphism (SNP) in a glutamate receptor-like gene (GhGLR4.8) on chromosome D03 was previously identified and validated to confer resistance to FOV race 7, and targeted genome sequencing demonstrated that it was also associated with resistance to FOV4. The objective of this study was to develop an easy and convenient PCR-based marker assay. To target the resistance SNP, a forward primer for the SNP with a mismatch in the 3rd position was designed for both the resistance (R) and susceptibility (S) alleles, respectively, with addition of 20-mer T7 promoter primer to the 5' end of the forward primer for the R allele. The two forward primers, in combination with each of five common reverse primers, were targeted to amplify amplicons of 50-260 bp in size with R and S alleles differing in 20 bp. Results showed that each of three common reverse primers in combination with the two forward primers produced polymorphic markers between R and S plants that were consistent with the targeted genome sequencing results. The polymorphism was distinctly resolved using both polyacrylamide and agarose gel electrophoreses. In addition, a sequence comparative analysis between the resistance gene and homologous sequences in sequenced tetraploid and diploid A and D genome species showed that none of the species possessed the resistance gene allele, suggesting its recent origin from a natural point mutation. The allele-specific PCR-based SNP typing method based on a three-primer combination provides a fast and convenient marker-assisted selection method to search and select for FOV4-resistant Upland cotton.

The Verticillium dahliae Small Cysteine-Rich Protein VdSCP23 Manipulates Host Immunity.

Verticillium wilt caused by Verticillium dahliae is a notorious soil-borne fungal disease and seriously threatens the yield of economic crops worldwide. During host infection, V. dahliae secretes many effectors that manipulate host immunity, among which small cysteine-rich proteins (SCPs) play an important role. However, the exact roles of many SCPs from V. dahliae are unknown and varied. In this study, we show that the small cysteine-rich protein VdSCP23 inhibits cell necrosis in Nicotiana benthamiana leaves, as well as the reactive oxygen species (ROS) burst, electrolyte leakage and the expression of defense-related genes. VdSCP23 is mainly localized in the plant cell plasma membrane and nucleus, but its inhibition of immune responses was independent of its nuclear localization. Site-directed mutagenesis and peptide truncation showed that the inhibition function of VdSCP23 was independent of cysteine residues but was dependent on the N-glycosylation sites and the integrity of VdSCP23 protein structure. Deletion of VdSCP23 did not affect the growth and development of mycelia or conidial production in V. dahliae. Unexpectedly, VdSCP23 deletion strains still maintained their virulence for N. benthamiana, Gossypium hirsutum and Arabidopsis thaliana seedlings. This study demonstrates an important role for VdSCP23 in the inhibition of plant immune responses; however, it is not required for normal growth or virulence in V. dahliae.

Durable and rechargeable antimicrobial cotton driven by enhanced UV stability and real-time detection of biocidal factors.

In this study, graphene oxide/N-halamine nanocomposite was synthesized through Pickering miniemulsion polymerization, which was then coated on cotton surface. The modified cotton exhibited excellent superhydrophobicity, which could effectively prevent microbial infestation and reduce the probability of hydrolysis of active chlorine, with virtually no active chlorine released in water after 72 h. Deposition of reduced graphene oxide nanosheets endowed cotton with ultraviolet-blocking properties, attributing to enhanced UV adsorption and long UV paths. Moreover, encapsulation of polymeric N-halamine resulted in improved UV stability, thus extending the life of N-halamine-based agents. After 24 h of irradiation, 85 % of original biocidal component (active chlorine content) was retained, and approximately 97 % of initial chlorine could be regenerated. Modified cotton has been proven to be an effective oxidizing material against organic pollutants and a potential antimicrobial substance. Inoculated bacteria were completely killed after 1 and 10 min of contact time, respectively. An innovative and simple scheme for determination of active chlorine content was also devised, and real-time inspection of bactericidal activity could be achieved to assure antimicrobial sustainability. Moreover, this method could be utilized to evaluate hazard classification of microbial contamination in different locations, thus broadening the application scope of N-halamine-based cotton fabrics.

Targeted development of diagnostic SNP markers for resistance to Fusarium wilt race 4 in Upland cotton (Gossypium hirsutum).

Fusarium wilt caused by the soil-borne fungus Fusarium oxysporum f. sp. vasinfectum (FOV) race 4 (FOV4) has become one of the most important emerging diseases in US cotton production. Numerous QTLs have been reported for resistance to FOV; however, no major FOV4-resistance QTL or gene has been identified and used in breeding Upland cotton (Gossypium hirsutum) for FOV4 resistance. In this study, a panel of 223 Chinese Upland cotton accessions was evaluated for FOV4 resistance based on seedling mortality rate (MR) and stem and root vascular discoloration (SVD and RVD). SNP markers were developed based on targeted genome sequencing using AgriPlex Genomics. The chromosome region at 2.130-2.292 Mb on D03 was significantly correlated with both SVD and RVD but not with MR. Based on the two most significant SNP markers, accessions homozygous for AA or TT SNP genotype averaged significantly lower SVD (0.88 vs. 2.54) and RVD (1.46 vs. 3.02) than those homozygous for CC or GG SNP genotype. The results suggested that a gene or genes within the region conferred resistance to vascular discoloration caused by FOV4. The Chinese Upland accessions had 37.22% homozygous AA or TT SNP genotype and 11.66% heterozygous AC or TG SNP genotype, while 32 US elite public breeding lines all had the CC or GG SNP genotype. Among 463 obsolete US Upland accessions, only 0.86% possessed the AA or TT SNP genotype. This study, for the first time, has developed diagnostic SNPs for marker-assisted selection and identified FOV4-resistant Upland germplasms with the SNPs.

Gene B5 in Cotton Confers High and Broad Resistance to Bacterial Blight and Conditions High Amounts of Sesquiterpenoid Phytoalexins.

Bacterial blight resistance gene B5 has received little attention since it was first described in 1950. A near-isogenic line (NIL) of Gossypium hirsutum cotton, AcB5, was generated in an otherwise bacterial-blight-susceptible 'Acala 44' background. The introgressed locus B5 in AcB5 conferred strong and broad-spectrum resistance to bacterial blight. Segregation patterns of test crosses under Oklahoma field conditions indicated that AcB5 is likely homozygous for resistance at two loci with partial dominance gene action. In controlled-environment conditions, two of the four copies of B5 were required for effective resistance. Contrary to expectations of gene-for-gene theory, AcB5 conferred high resistance toward isogenic strains of Xanthomonas citri subsp. malvacearum carrying cloned avirulence genes avrB4, avrb7, avrBIn, avrB101, and avrB102, respectively, and weaker resistance toward the strain carrying cloned avrb6. The hypothesis that each B gene, in the absence of a polygenic complex, triggers sesquiterpenoid phytoalexin production was tested by measurement of cadalene and lacinilene phytoalexins during resistant responses in five NILs carrying different B genes, four other lines carrying multiple resistance genes, as well as susceptible Ac44E. Phytoalexin production was an obvious, but variable, response in all nine resistant lines. AcB5 accumulated an order of magnitude more of all four phytoalexins than any of the other resistant NILs. Its total levels were comparable to those detected in OK1.2, a highly resistant line that possesses several B genes in a polygenic background.

GbCYP72A1 Improves Resistance to Verticillium Wilt via Multiple Signaling Pathways.

Verticillium dahliae is a fungal pathogen that causes Verticillium wilt (VW), which seriously reduces the yield of cotton owing to biological stress. The mechanism underlying the resistance of cotton to VW is highly complex, and the resistance breeding of cotton is consequently limited by the lack of in-depth research. Using quantitative trait loci (QTL) mapping, we previously identified a novel cytochrome P450 (CYP) gene on chromosome D4 of Gossypium barbadense that is associated with resistance to the nondefoliated strain of V. dahliae. In this study, the CYP gene on chromosome D4 was cloned together with its homologous gene on chromosome A4 and were denoted as GbCYP72A1d and GbCYP72A1a, respectively, according to their genomic location and protein subfamily classification. The two GbCYP72A1 genes were induced by V. dahliae and phytohormone treatment, and the findings revealed that the VW resistance of the lines with silenced GbCYP72A1 genes decreased significantly. Transcriptome sequencing and pathway enrichment analyses revealed that the GbCYP72A1 genes primarily affected disease resistance via the plant hormone signal transduction, plant-pathogen interaction, and mitogen-activated protein kinase (MAPK) signaling pathways. Interestingly, the findings revealed that although GbCYP72A1d and GbCYP72A1a had high sequence similarity and both genes enhanced the disease resistance of transgenic Arabidopsis, there was a difference between their disease resistance abilities. Protein structure analysis revealed that this difference was potentially attributed to the presence of a synaptic structure in the GbCYP72A1d protein. Altogether, the findings suggested that the GbCYP72A1 genes play an important role in plant response and resistance to VW.

Effect of volatile compounds produced by the cotton endophytic bacterial strain Bacillus sp. T6 against Verticillium wilt.

BACKGROUND: Verticillium wilt, caused by the fungus Verticillium dahliae, leads to significant losses in cotton yield worldwide. Biocontrol management is a promising means of suppressing verticillium wilt. The purpose of the study was to obtain and analyze endophytic bacteria with Verticillium wilt-resistant activities from the roots of Gossypium barbadense 'Xinhai15' and to explore the interactions between the soil and plants. RESULTS: An endophytic bacterium Bacillus sp. T6 was obtained from the Verticillium wilt-resistant cotton G. barbadense 'Xinhai15', which showed significant antagonistic abilities against cotton Verticillium wilt. The bioassay results indicated that the strain possessed strong antagonistic abilities that inhibited V. dahliae spore germination and mycelial growth without contact, and thus it was speculated that the active factor of the bacteria might be volatile compounds. A total of 46 volatile substances were detected via headspace solid-phase microextraction and gas chromatography-mass spectrometry analysis. The pure product verification experiment confirmed that the styrene produced by the T6 strain was the main virulence factor. Transcriptome analysis showed that following styrene induction, 247 genes in V. dahliae, including four hydrolase genes, eight dehydrogenase genes, 11 reductase genes, 17 genes related to transport and transfer were upregulated. Additionally, 72 genes, including two chitinase genes, two protease genes, five transport-related genes, and 33 hypothetical protein genes, were downregulated. The quantitative real-time PCR results confirmed that the expression of the four genes VDAG_02838, VDAG_09554, VDAG_045572, and VDAG_08251 was increased by 3.18, 78.83, 2.71, and 2.92 times, respectively, compared with the uninduced control group. CONCLUSIONS: The research provides a new reference for the development and application of the volatile compounds of endophytic bacteria as new biocontrol agents for the control of Verticillium wilt and as biological preservatives for agricultural products.

Genome Resource for the Verticillium Wilt Resistant Gossypium hirsutum Cultivar Zhongzhimian No. 2.

Verticillium wilt, caused by the fungal pathogen Verticillium dahliae, is the major cause of disease-related yield losses in cotton (Gossypium hirsutum). Despite these losses, the major cultivars of G. hirsutum remain highly susceptible to Verticillium wilt. The lack of understanding on the genetic basis for Verticillium wilt resistance may further hinder progress in deploying elite cultivars with proven resistance, such as the wilt resistant G. hirsutum cultivar Zhongzhimian No. 2. To help remedy this knowledge gap, we sequenced the whole genome of Zhongzhimian No. 2 and assembled it from a combination of PacBio long reads, Illumina short reads, and high-throughput chromosome conformation capture technologies. The final assembly of the genome was 2.33 Gb, encoding 95,327 predicted coding sequences. The GC content was 34.39% with 99.2% of the bases anchored to 26 pseudo-chromosomes that ranged from 53.8 to 127.7 Mb. This resource will help gain a detailed understanding of the genomic features governing high yield and Verticillium wilt resistance in this cultivar. Comparative genomics will be particularly helpful, since there are several published genomes of other Gossypium species. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Response of the plant core microbiome to Fusarium oxysporum infection and identification of the pathobiome.

Plant core microbiomes consist of persistent key members that provide critical host functions, but their assemblages can be interrupted by biotic and abiotic stresses. The pathobiome is comprised of dynamic microbial interactions in response to disease status of the host. Hence, identifying variation in the core microbiome and pathobiome can significantly advance our understanding of microbial-microbial interactions and consequences for disease progression and host functions. In this study, we combined glasshouse and field studies to analyse the soil and plant rhizosphere microbiome of cotton plants (Gossypium hirsutum) in the presence of a cotton-specific fungal pathogen, Fusarium oxysporum f. sp. vasinfectum (FOV). We found that FOV directly and consistently altered the rhizosphere microbiome, but the biocontrol agents enabled microbial assemblages to resist pathogenic stress. Using co-occurrence network analysis of the core microbiome, we identified the pathobiome comprised of the pathogen and key associate phylotypes in the cotton microbiome. Isolation and application of some negatively correlated pathobiome members provided protection against plant infection. Importantly, our field survey from multiple cotton fields validated the pattern and responses of core microbiomes under FOV infection. This study advances key understanding of core microbiome responses and existence of plant pathobiomes, which provides a novel framework to better manage plant diseases in agriculture and natural settings.

Effect of 1-aminocyclopropane-1-carboxylic acid accumulation on Verticillium dahliae infection of upland cotton.

BACKGROUND: Verticillium wilt of cotton is a serious disease caused by the infection of soil borne fungus Verticillium dahliae Kleb, and the infection mechanisms may involve the regulation of phytohormone ethylene. The precursor of ethylene biosynthesis is 1-aminocyclopropane-1-carboxylic acid (ACC), whose biosynthesis in vivo depends on activation of ACC synthase (ACS). Here, we investigated how ACS activation and ACC accumulation affected the infection of V. dahliae strain Vd991 on cotton (Gossypium hirsutum L.) cultivar YZ1. RESULTS: Preliminary observations indicated that ACC applications reduced the disease incidence, disease index and stem vascular browning by impeding fungal biomass accumulation. Transcriptome and qRT-PCR data disclosed that Vd991 induced GhACS2 and GhACS6 expression. GhACS2- or GhACS6-overexpressing transgenic YZ1 lines were generated, respectively. In a Verticillium disease nursery with about 50 microsclerotia per gram of soil, these ACC-accumulated plants showed decreased disease indexes, stem fungal biomasses and vascular browning. More importantly, these transgenic plants decreased the green fluorescent protein-marked Vd991 colonization and diffusion in root tissues. Further, either ACC treatment or ACC-accumulating cotton plants activated salicylic acid (SA)-dependent resistance responses. CONCLUSIONS: The GhACS2- and GhACS6-dependent ACC accumulations enhanced the resistance of cotton to V. dahliae in a SA-dependent manner, and this lays a foundation for cotton resistance breeding.

Identification and expression analysis of arabinogalactan protein genes in cotton reveal the function of GhAGP15 in Verticillium dahliae resistance.

Verticillium wilt, primarily caused by the fungal pathogen Verticillium dahliae, is a serious disease in cotton. Arabinogalactan proteins (AGPs), a class of hydroxyproline-rich glycoproteins, have been widely implicated in plant growth and environmental adaptation. The purpose of this study is to identify and characterize AGP members in cotton plants and explore their roles in responding to environmental stressors. In total, 65 GhAGP members were identified in upland cotton (Gossypium hirsutum), along with 43, 35, and 37 AGP members that were also identified in G. barbadense, G. arboreum, and G. raimondii, respectively. According to gene structure and protein domains analysis, GhAGP genes in upland cotton are highly conserved. Meanwhile, tandem duplication events have occurred frequently throughout cotton's evolutionary history. Expression analysis showed that GhAGP genes were widely expressed during growth and development and in response to abiotic stressors. Many cis-elements related to hormonal responses and environmental stressors were detected in GhAGP promoter regions. GhAGP genes participate in responding to cold, drought, and salt stress, and were sensitive to ET signaling. Furthermore, the expression level of GhAGP15 was elevated during V. dahliae infection and resistance against V. dahliae in upland cotton was significantly weakened by silencing GhAGP15 using a virus-induced gene silencing (VIGS) approach. Our results further suggest that the function of GhAGP15 in V. dahliae resistance might be involved in regulation of the JA, SA, and reactive oxygen species (ROS) pathways. The comprehensive analysis of AGP genes in cotton performed in this study provides a basic framework for further functional research of these genes.

Ramularia leaf spot and boll rot are affected differently by organic and inorganic nitrogen fertilization in cotton plants

Interaction among nitrogen fertilization using bovine manure, poultry manure, Jatropha curcas seed cake and urea, and the diseases Ramularia leaf spot (RLS) and Boll rot (BR), caused by Ramulariopsis pseudoglycines and Diplodia gossypina, respectively, in cotton plants (Gossypium hirsutum L.), was studied under field conditions. Intensity (incidence and severity in percentage) of RLS and incidence (%) of BR were evaluated over time, starting in reproductive stage B1 (first visible flower bud). A randomized complete block design with a 4x4 factorial arrangement was used (fertilizers x doses), totaling 16 treatments with four replications. Disease progress was analyzed with the nonlinear Logistic and Gompertz models, obtaining the epidemiological parameters amount of initial disease (Y0) and progress rate (r). Plants fertilized with 50 kg N ha-1, presented an incidence twice greater than those obtained with other fertilizers. The Logistic model better fits RLS, but no model could represent BR. Only the epidemiological parameters of RLS were affected differently in this experiment compared to BR disease. The possible role of organic and inorganic nitrogen fertilization in the RLS and BR management is discussed.

Mapping of dynamic QTLs for resistance to Fusarium wilt (Fusarium oxysporum f. sp. vasinfectum) race 4 in a backcross inbred line population of Upland cotton.

KEY MESSAGE: A backcross inbred line population of cotton was evaluated for Fusarium wilt race 4 resistance at different days after inoculation (DAI). Both constitutively expressed and developmentally regulated QTLs were detected. The soil-borne fungus Fusarium oxysporum f. sp. vasinfectum (FOV) race 4 (FOV4) causes Fusarium wilt including seedling mortality in cotton. A backcross inbred line (BIL) population of 181 lines, derived from a bi-parental cross of moderately resistant non-recurrent Hai 7124 (Gossypium barbadense) and recurrent parent CCRI 36 (G. hirsutum), was evaluated under temperature-controlled conditions for FOV4 resistance with artificial inoculations. Based on three replicated tests evaluated at 7, 14, 21, and 28 days after inoculation (DAI), only 2-5 BILs showed lower disease severity ratings (DSR) than the parents while 22-50 BILs were more susceptible, indicating transgressive segregation toward susceptibility. Although DSR were overall congruent between DAI, there were many BILs displaying different responses to FOV4 across DAI. Genetic mapping using 7709 SNP markers identified 42 unique QTLs for four evaluation parameters- disease incidence (DI), DSR, mortality rate (MR), and area under disease progress curve (AUDPC), including 26 for two or more parameters. All five QTLs for AUDPC were co-localized with QTLs for DI, DSR, and/or MR at one or two DAI, indicating the unnecessary use of AUDPC in QTL mapping for FOV4 resistance. Those common QTLs explained the significant positive associations between parameters observed. Ten common QTLs with negative or positive additive effects were detected between DAI. DAI-specific and consistent QTLs were detected between DAI in cotton for the first time, suggesting the existence of both constitutively expressed and developmentally regulated QTLs for FOV4 resistance and the importance of evaluating genetic populations for FOV4 resistance at different growth stages.

DNA-Based Quantification of Fusarium oxysporum f. sp. vasinfectum in Environmental Soils to Describe Spatial Variation in Inoculum Density.

Fusarium wilt of cotton, caused by the soilborne fungal pathogen Fusarium oxysporum f. sp. vasinfectum (FOV), occurs in regions of the United States where cotton (Gossypium spp.) is grown. Race 4 of this pathogen (FOV4) is especially aggressive, and does not require the co-occurrence of the root knot nematode (Meloidogyne incognita) to infect cotton. Its sudden appearance in far-west Texas in 2016 after many years of being restricted to California is of great concern, as is the threat of its continued spread through the cotton-producing regions of the United States. The aim of this research was to analyze the spatial variability of FOV4 inoculum density in the location where FOV4 is locally emerging, using quantitative and droplet digital PCR methods. Soil samples collected from a field with known FOV4 incidence in Fabens, Texas, were analyzed. Appreciable variation in inoculum density was found to occur at spatial scales smaller than the size of plots involved in cultivar trial research, and was spatially autocorrelated (Moran's I, Z = 17.73, P < 0.0001). These findings indicate that, for cultivar trials, accounting for the spatial distribution of inoculum, either by directly quantifying it or through the use of densely distributed calibration checks, is important to the interpretation of results.

Identification of long non-coding RNAs in Verticillium dahliae following inoculation of cotton.

Long non-coding RNAs (lncRNAs) play important roles in diverse biological processes. However, these functions have not been assessed in Verticillium dahliae, a soil-borne fungal pathogen that causes devastating wilt diseases in many crops. The discovery and identity of novel lncRNAs and their association with virulence may contribute to an increased understanding of the regulation of virulence in V. dahliae. Here, we identified a total of 352 lncRNAs in V. dahliae. The lncRNAs were transcribed from all V. dahliae chromosomes, typically with shorter open reading frames, lower GC content, and fewer exons than protein-coding genes. In addition, 308 protein-coding genes located within 10 kb upstream and 10 kb downstream of lncRNAs were identified as neighboring genes, and which were considered as potential targets of lncRNA. These neighboring genes encode products involved in development, stress responses, and pathogenicity of V. dahliae, such as transcription factors (TF), kinase, and members of the secretome. Furthermore, 47 lncRNAs were significantly differentially expressed in V. dahliae following inoculation of susceptible cotton (Gossyoiumhisutum) cultivar Junmian No.1, suggesting that lncRNAs may be involved in the regulation of virulence in V. dahliae. Moreover, correlations in expression patterns between lncRNA and their neighboring genes were detected. Expression of lncRNA012077 and its neighboring gene was up-regulated 6 h following inoculation of cotton, while the expression of lncRNA007722 was down-regulated at 6 h but up-regulated at 24 h, in a pattern opposite to that of its neighboring gene. Overexpression of lncRNA012077 in wild-type strain (Vd991) enhanced its virulence on cotton while overexpression of lncRNA009491 reduced virulence. Identification of novel lncRNAs and their association with virulence may provide new targets for disease control.

Long Non-Coding RNAs profiling in pathogenesis of Verticillium dahliae: New insights in the host-pathogen interaction.

Verticillium dahliae causes vascular wilt disease on cotton (Gossypium hirsutum), resulting in devastating yield loss worldwide. While little is known about the mechanism of long non-coding RNAs (lncRNAs), several lncRNAs have been implicated in numerous physiological processes and diseases. To better understand V. dahliae pathogenesis, lncRNA was conducted in a V. dahliae virulence model. Potential target genes of significantly regulated lncRNAs were predicted using cis/trans-regulatory algorithms. This study provides evidence for lncRNAs' regulatory role in pathogenesis-related genes. Interestingly, lncRNAs were identified and varying in terms of RNA length and nutrient starvation treatments. Efficient pathogen nutrition during the interaction with the host is a requisite factor during infection. Our observations directly link to mutated V. dahliae invasion, explaining infected cotton have lower pathogenicity and lethality compared to V. dahliae. Remarkably, lncRNAs XLOC_006536 and XLOC_000836 involved in the complex regulation of pathogenesis-related genes in V. dahliae were identified. For the first time the regulatory role of lncRNAs in filamentous fungi was uncovered, and it is our contention that elucidation of lncRNAs will advance our understanding in the development and pathogenesis of V. dahliae and offer alternatives in the control of the diseases caused by fungus V. dahliae attack.

Silencing GhIAA43, a member of cotton AUX/IAA genes, enhances wilt resistance via activation of salicylic acid-mediated defenses.

Auxin-mediated degradation of Aux/IAA proteins is a crucial step in auxin signaling. Recent researches indicate that Aux/IAA members also play a role in biotic and abiotic stresses. For example, Pseudomonas syringae infection causes Arabidopsis Aux/IAA protein (AXR2, AXR3) turnover. Here, by analyzing RNA-seq data we found that several cotton Aux/IAA genes are responsive to Verticillium dahliae infection, one of these named GhIAA43, was investigated for its role in cotton defense against V. dahliae infection. We demonstrate that the transcript levels of GhIAA43 were responsive to both V. dahliae infection and exogenous IAA application. By producing transgenic Arabidopsis plants overexpressing GhIAA43-GUS fusion, we show that IAA treatment and V. dahliae infection promoted GhIAA43 protein turnover. Silencing GhIAA43 in cotton enhanced wilt resistance, suggesting that GhIAA43 is a negative regulator in cotton defense against V. dahliae attack. By monitoring SA marker gene expression and measurement of SA content in GhIAA43-silenced cotton plants, we found that the enhanced resistance in GhIAA43-silenced cotton plants is due to the activation of SA-related defenses, and the activated defenses specifically occurred in the presence of V. dahliae. Furthermore, exogenous IAA application improve wilt resistance in cotton plants tested. Our results provide novel connection between auxin signaling and SA-related defenses in cotton upon V. dahliae attack.

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.

An In Vitro Co-Culture System for Rapid Differential Response to Fusarium oxysporum f. sp. vasinfectum Race 4 in Three Cotton Cultivars.

Fusarium oxysporum f. sp. vasinfectum race 4 (FOV4) is a devastating fungus pathogen that causes Fusarium wilt in both domesticated cotton species, Gossypium hirsutum (Upland) and G. barbadense (Pima). Greenhouse and field-based pathogenicity assays can be a challenge because of nonuniform inoculum levels, the presence of endophytes, and varying environmental factors. Therefore, an in vitro coculture system was designed to support the growth of both domesticated cotton species and FOV4 via an inert polyphenolic foam substrate with a liquid medium. A Fusarium wilt-susceptible Pima cotton cultivar, G. barbadense 'GB1031'; a highly resistant Pima cotton cultivar, G. barbadense 'DP348RF'; and a susceptible Upland cotton cultivar, G. hirsutum 'TM-1', were evaluated for 30 days during coculture with FOV4 in this foam-based system. Thirty days after inoculation, disease symptoms were more severe in both susceptible cultivars, which displayed higher percentages of foliar damage, and greater plant mortality than observed in 'DP348RF', the resistant Pima cotton cultivar. This foam-based in vitro system may be useful for screening cotton germplasm for resistance to a variety of fungus pathogens and may facilitate the study of biotic interactions in domesticated cotton species under controlled environmental conditions.