Positive roles of the Ca2+ sensors GbCML45 and GbCML50 in improving cotton Verticillium wilt resistance.

As a universal second messenger, cytosolic calcium (Ca2+) functions in multifaceted intracellular processes, including growth, development and responses to biotic/abiotic stresses in plant. The plant-specific Ca2+ sensors, calmodulin and calmodulin-like (CML) proteins, function as members of the second-messenger system to transfer Ca2+ signal into downstream responses. However, the functions of CMLs in the responses of cotton (Gossypium spp.) after Verticillium dahliae infection, which causes the serious vascular disease Verticillium wilt, remain elusive. Here, we discovered that the expression level of GbCML45 was promoted after V. dahliae infection in roots of cotton, suggesting its potential role in Verticillium wilt resistance. We found that knockdown of GbCML45 in cotton plants decreased resistance while overexpression of GbCML45 in Arabidopsis thaliana plants enhanced resistance to V. dahliae infection. Furthermore, there was physiological interaction between GbCML45 and its close homologue GbCML50 by using yeast two-hybrid and bimolecular fluorescence assays, and both proteins enhanced cotton resistance to V. dahliae infection in a Ca2+-dependent way in a knockdown study. Detailed investigations indicated that several defence-related pathways, including salicylic acid, ethylene, reactive oxygen species and nitric oxide signalling pathways, as well as accumulations of lignin and callose, are responsible for GbCML45- and GbCML50-modulated V. dahliae resistance in cotton. These results collectively indicated that GbCML45 and GbCML50 act as positive regulators to improve cotton Verticillium wilt resistance, providing potential targets for exploitation of improved Verticillium wilt-tolerant cotton cultivars by genetic engineering and molecular breeding.

Transcriptome analysis of Gossypium hirsutum cultivar Zhongzhimian No.2 uncovers the gene regulatory networks involved in defense against Verticillium dahliae.

BACKGROUND: Cotton is globally important crop. Verticillium wilt (VW), caused by Verticillium dahliae, is the most destructive disease in cotton, reducing yield and fiber quality by over 50% of cotton acreage. Breeding resistant cotton cultivars has proven to be an efficient strategy for improving the resistance of cotton to V. dahliae. However, the lack of understanding of the genetic basis of VW resistance may hinder the progress in deploying elite cultivars with proven resistance. RESULTS: We planted the VW-resistant Gossypium hirsutum cultivar Zhongzhimian No.2 (ZZM2) in an artificial greenhouse and disease nursery. ZZM2 cotton was subsequently subjected to transcriptome sequencing after Vd991 inoculation (6, 12, 24, 48, and 72 h post-inoculation). Several differentially expressed genes (DEGs) were identified in response to V. dahliae infection, mainly involved in resistance processes, such as flavonoid and terpenoid quinone biosynthesis, plant hormone signaling, MAPK signaling, phenylpropanoid biosynthesis, and pyruvate metabolism. Compared to the susceptible cultivar Junmian No.1 (J1), oxidoreductase activity and reactive oxygen species (ROS) production were significantly increased in ZZM2. Furthermore, gene silencing of cytochrome c oxidase subunit 1 (COX1), which is involved in the oxidation-reduction process in ZZM2, compromised its resistance to V. dahliae, suggesting that COX1 contributes to VW resistance in ZZM2. CONCLUSIONS: Our data demonstrate that the G. hirsutum cultivar ZZM2 responds to V. dahliae inoculation through resistance-related processes, especially the oxidation-reduction process. This enhances our understanding of the mechanisms regulating the ZZM2 defense against VW.

SR45a plays a key role in enhancing cotton resistance to Verticillium dahliae by alternative splicing of immunity genes.

Alternative splicing (AS) of pre-mRNAs increases the diversity of transcriptome and proteome and plays fundamental roles in plant development and stress responses. However, the prevalent changes in AS events and the regulating mechanisms of plants in response to pathogens remain largely unknown. Here, we show that AS changes are an important mechanism conferring cotton immunity to Verticillium dahliae (Vd). GauSR45a, encoding a serine/arginine-rich RNA binding protein, was upregulated expression and underwent AS in response to Vd infection in Gossypium australe, a wild diploid cotton species highly resistant to Vd. Silencing GauSR45a substantially reduced the splicing ratio of Vd-induced immune-associated genes, including GauBAK1 (BRI1-associated kinase 1) and GauCERK1 (chitin elicitor receptor kinase 1). GauSR45a binds to the GAAGA motif that is commonly found in the pre-mRNA of genes essential for PTI, ETI, and defense. The binding between GauSR45a and the GAAGA motif in the pre-mRNA of BAK1 was enhanced by two splicing factors of GauU2AF35B and GauU1-70 K, thereby facilitating exon splicing; silencing either AtU2AF35B or AtU1-70 K decreased the resistance to Vd in transgenic GauSR45a Arabidopsis. Overexpressing the short splicing variant of BAK1GauBAK1.1 resulted in enhanced Verticillium wilt resistance rather than the long one GauBAK1.2. Vd-induced far more AS events were in G. barbadense (resistant tetraploid cotton) than those in G. hirsutum (susceptible tetraploid cotton) during Vd infection, indicating resistance divergence in immune responses at a genome-wide scale. We provided evidence showing a fundamental mechanism by which GauSR45a enhances cotton resistance to Vd through global regulation of AS of immunity genes.

Promotion of apoplastic oxidative burst by artificially selected GhCBSX3A enhances Verticillium dahliae resistance in upland cotton.

Verticillium wilt (VW) is a devasting disease affecting various plants, including upland cotton, a crucial fiber crop. Despite its impact, the genetic basis underlying cotton's susceptibility or defense against VW remains unclear. Here, we conducted a genome-wide association study on VW phenotyping in upland cotton and identified a locus on A13 that is significantly associated with VW resistance. We then identified a cystathionine ß-synthase domain gene at A13 locus, GhCBSX3A, which was induced by Verticillium dahliae. Functional analysis, including expression silencing in cotton and overexpression in Arabidopsis thaliana, confirmed that GhCBSX3A is a causal gene at the A13 locus, enhancing SAR-RBOHs-mediated apoplastic oxidative burst. We found allelic variation on the TATA-box of GhCBSX3A promoter attenuated its expression in upland cotton, thereby weakening VW resistance. Interestingly, we discovered that altered artificial selection of GhCBSX3A_R (an elite allele for VW) under different VW pressures during domestication and other improved processes allows specific human needs to be met. Our findings underscore the importance of GhCBSX3A in response to VW, and we propose a model for defense-associated genes being selected depending on the pathogen's pressure. The identified locus and gene serve as promising targets for VW resistance enhancement in cotton through genetic engineering.

The Protein Phosphatase GhAP2C1 Interacts Together with GhMPK4 to Synergistically Regulate the Immune Response to Fusarium oxysporum in Cotton.

The plant mitogen-activated protein kinase (MAPK) cascade plays an important role in mediating responses to biotic and abiotic stresses and is the main pathway through which extracellular stimuli are transduced intracellularly as signals. Our previous research showed that the GhMKK6-GhMPK4 cascade signaling pathway plays an important role in cotton immunity. To further analyze the role and regulatory mechanism of the GhMKK6-GhMPK4 cascade signaling pathway in cotton resistance to Fusarium wilt, we functionally analyzed GhMPK4. Our results show that silencing GhMPK4 reduces cotton tolerance to Fusarium wilt and reduces the expression of several resistance genes. Further experiments revealed that GhMPK4 is similar to GhMKK6, both of whose overexpression cause unfavorable cotton immune response characteristics. By using a yeast two-hybrid screening library and performing a bioinformatics analysis, we screened and identified a negative regulator of the MAPK kinase-protein phosphatase AP2C1. Through the functional analysis of AP2C1, it was found that, after being silenced, GhAP2C1 increased resistance to Fusarium wilt, but GhAP2C1 overexpression caused sensitivity to Fusarium wilt. These findings show that GhAP2C1 interacts together with GhMPK4 to regulate the immune response of cotton to Fusarium oxysporum, which provides important data for functionally analyzing and studying the feedback regulatory mechanism of the MAPK cascade and helps to clarify the regulatory mechanism through which the MAPK cascade acts in response to pathogens.

Analysis of a tetraploid cotton line Mac7 transcriptome reveals mechanisms underlying resistance against the whitefly Bemisia tabaci.

Whitefly inflicts both direct and indirect losses to cotton crop. Whitefly resistant cotton germplasm is a high priority and considered among the best possible solutions to mitigate this issue. In this study, we evaluated cotton leaf curl disease (CLCuD) resistant cotton line Mac7 under whitefly stress. Furthermore, we utilized the already available transcriptome data of Mac7 concerning whitefly stress to elucidate associated mechanisms and identify functionally important genes in cotton. In transcriptomic data analysis, differentially expressed genes (DEGs) were found involved in complex relay pathways, activated on whitefly exposure. The response implicates signalling through resistance genes (R-genes), MAPK, ROS, VQs or RLKs, transcription factors, which leads to the activation of defence responses including, Ca2+messengers, phytohormonal cross-talk, gossypol, flavonoids, PhasiRNA and susceptibility genes (S-genes). The qRT-PCR assay of 10 functionally important genes also showed their involvement in differential responses at 24 and 48 h post whitefly infestation. Briefly, our study helps in understanding the resistant nature of Mac7 under whitefly stress.

Evolutionary and Characteristic Analysis of RING-DUF1117 E3 Ubiquitin Ligase Genes in Gossypium Discerning the Role of GhRDUF4D in Verticillium dahliae Resistance.

Verticillium wilt, primarily induced by the soil-borne fungus Verticillium dahliae, is a serious threat to cotton fiber production. There are a large number of really interesting new gene (RING) domain-containing E3 ubiquitin ligases in Arabidopsis, of which three (At2g39720 (AtRHC2A), At3g46620 (AtRDUF1), and At5g59550 (AtRDUF2)) have a domain of unknown function (DUF) 1117 domain in their C-terminal regions. This study aimed to detect and characterize the RDUF members in cotton, to gain an insight into their roles in cotton's adaptation to environmental stressors. In this study, a total of 6, 7, 14, and 14 RDUF (RING-DUF1117) genes were detected in Gossypium arboretum, G. raimondii, G. hirsutum, and G. barbadense, respectively. These RDUF genes were classified into three groups. The genes in each group were highly conserved based on gene structure and domain analysis. Gene duplication analysis revealed that segmental duplication occurred during cotton evolution. Expression analysis revealed that the GhRDUF genes were widely expressed during cotton growth and under abiotic stresses. Many cis-elements related to hormone response and environment stressors were identified in GhRDUF promoters. The predicted target miRNAs and transcription factors implied that GhRDUFs might be regulated by gra-miR482c, as well as by transcription factors, including MYB, C2H2, and Dof. The GhRDUF genes responded to cold, drought, and salt stress and were sensitive to jasmonic acid, salicylic acid, and ethylene signals. Meanwhile, GhRDUF4D expression levels were enhanced after V. dahliae infection. Subsequently, GhRDUF4D was verified by overexpression in Arabidopsis and virus-induced gene silencing treatment in upland cotton. We observed that V. dahliae resistance was significantly enhanced in transgenic Arabidopsis, and weakened in GhRDUF4D silenced plants. This study conducted a comprehensive analysis of the RDUF genes in Gossypium, hereby providing basic information for further functional studies.

Identification of cell wall-associated kinases as important regulators involved in Gossypium hirsutum resistance to Verticillium dahliae.

BACKGROUND: Verticillium wilt, caused by the soil borne fungus Verticillium dahliae, is a major threat to cotton production worldwide. An increasing number of findings indicate that WAK genes participate in plant-pathogen interactions, but their roles in cotton resistance to V. dahliae remain largely unclear. RESULTS: Here, we carried out a genome-wide analysis of WAK gene family in Gossypium hirsutum that resulted in the identification of 81 putative GhWAKs, which were all predicated to be localized on plasma membrane. In which, GhWAK77 as a representative was further located in tobacco epidermal cells using transient expression of fluorescent fusion proteins. All GhWAKs could be classified into seven groups according to their diverse protein domains, indicating that they might sense different outside signals to trigger intracellular signaling pathways that were response to various environmental stresses. A lot of cis-regulatory elements were predicted in the upstream region of GhWAKs and classified into four main groups including hormones, biotic, abiotic and light. As many as 28 GhWAKs, playing a potential role in the interaction between cotton and V. dahliae, were screened out by RNA-seq and qRT-PCR. To further study the function of GhWAKs in cotton resistance to V. dahliae, VIGS technology was used to silence GhWAKs. At 20 dpi, VIGSed plants exhibited more chlorosis and wilting than the control plants. The disease indices of VIGSed plants were also significantly higher than those of the control. Furthermore, silencing of GhWAKs significantly affected the expression of JA- and SA-related marker genes, increased the spread of V. dahliae in the cotton stems, dramatically compromised V. dahliae-induced accumulation of lignin, H2O2 and NO, but enhanced POD activity. CONCLUSION: Our study presents a comprehensive analysis on cotton WAK gene family for the first time. Expression analysis and VIGS assay provided direct evidences on GhWAKs participation in the cotton resistance to V. dahliae.

GhWRKY1-like enhances cotton resistance to Verticillium dahliae via an increase in defense-induced lignification and S monolignol content.

Cotton is one of the most important economic crops and is cultivated globally. Verticillium wilt, caused by the soil-borne hemibiotrophic fungus Verticillium dahliae, is the most destructive disease in cotton production for its infection strategies and great genetic plasticity. Recent studies have identified the accumulation of lignin is a general and basal defense reaction in plant immunity and cotton resistance to V. dahliae. However, the functions and regulatory mechanisms of transcription factors in cotton defense-induced lignification and lignin composition alteration were less reported. Here, we identified a WRKY transcription factor GhWRKY1-like from upland cotton (Gossypium hirsutum) as a positive regulator in resistance to V. dahliae via directly manipulating lignin biosynthesis. Further analysis revealed that GhWRKY1-like interacts with the promoters of lignin biosynthesis related genes GhPAL6 and GhCOMT1, and activates the expression of GhPAL6 and GhCOMT1, which led to enhanced total lignin especially S monomers biosynthesis. These results demonstrate that GhWRKY1-like enhances Verticillium wilt resistance via an increase in defense-induced lignification and broaden our knowledge of the roles of lignification and the lignin composition in plant defense responses.

GhVLN4 is involved in multiple stress responses and required for resistance to Verticillium wilt.

As structural and signaling platform in plant cell, the actin cytoskeleton is regulated by diverse actin binding proteins (ABPs). Villins are one type of major ABPs responsible for microfilament bundling, which have proved to play important roles in plant growth and development. However, the function of villins in stress tolerance is poorly understood. Here, we report the function of cotton GhVLN4 in Verticillium wilt resistance and abiotic stress tolerance. The expression of GhVLN4 was up-regulated by gibberellin, ethylene, ABA, salicylic acid, jasmonate, NaCl, PEG, and Verticillium dahliae treatment, suggesting the involvement of GhVLN4 in multiple stress and hormone responses and signaling. Virus-induced gene silencing GhVLN4 made cotton more susceptible to V. dahliae characterized by the preferential colonization and rapid growth of the fungus in both phloem and xylem of the infected stems. Arabidopsis overexpressing GhVLN4 exhibited higher resistance to V. dahliae, salt and drought than the wild-type plants. The enhanced resistance to V. dahliae is likely related to the upregulated components in SA signaling pathway; the improved tolerance to salt and drought is characterized by upregulation of the components both in ABA- related and ABA-independent signal pathways, along with altered stomatal aperture under drought. Our findings demonstrate that GhVLN4 may play important roles in regulating plant tolerance to both biotic and abiotic stresses.

Phosphate deficiency enhances cotton resistance to Verticillium dahliae through activating jasmonic acid biosynthesis and phenylpropanoid pathway.

Living in natural environment, plants often suffer from various biotic and abiotic stresses. Phosphate deficiency is a common factor affecting crop production in field, while pathogen invasion is another serious problem. Here we report that Pi-deficient cotton plants exhibit enhanced resistance to Verticillium dahliae. Transcriptomic and histochemical analysis revealed that cotton phenylpropanoid pathway was activated under phosphate deficiency, including lignin and flavonoid biosynthesis. Metabolomic data showed that Pi-deficient cotton accumulates many flavonoids metabolites and displays obvious anti-fungi activity in terms of methanolic extract. Additionally, JA biosynthesis was activated under phosphate deficiency and the Pi-deficiency induced disease resistance was significantly attenuated in GhAOS knock down plants. Taken together, our study demonstrated that phosphate deficiency enhanced cotton resistance to V. dahliae through activating phenylpropanoid pathway and JA biosynthesis, providing new insights into how phosphate deficiency affects plant disease resistance.

Genome-Wide Analysis of OPR Family Genes in Cotton Identified a Role for GhOPR9 in Verticillium dahliae Resistance.

The 12-oxo-phytodienoic acid reductases (OPRs) have been proven to play a major role in plant development and growth. Although the classification and functions of OPRs have been well understood in Arabidopsis, tomato, rice, maize, and wheat, the information of OPR genes in cotton genome and their responses to biotic and abiotic stresses have not been reported. In this study, we found 10 and 9 OPR genes in Gossypium hirsutum and Gossypium barbadense, respectively. They were classified into three groups, based on the similar gene structure and conserved protein motifs. These OPR genes just located on chromosome 01, chromosome 05, and chromosome 06. In addition, the whole genome duplication (WGD) or segmental duplication events contributed to the evolution of the OPR gene family. The analyses of cis-acting regulatory elements of GhOPRs showed that the functions of OPR genes in cotton might be related to growth, development, hormone, and stresses. Expression patterns showed that GhOPRs were upregulated under salt treatment and repressed by polyethylene glycol 6000 (PEG6000). The expression patterns of GhOPRs were different in leaf, root, and stem under V. dahliae infection. GhOPR9 showed a higher expression level than other OPR genes in cotton root. The virus-induced gene silencing (VIGS) analysis suggested that knockdown of GhOPR9 could increase the susceptibility of cotton to V. dahliae infection. Furthermore, GhOPR9 also modulated the expressions of jasmonic acid (JA) pathway-regulated genes under the V. dahliae infection. Overall, our results provided the evolution and potential functions of the OPR genes in cotton. These findings suggested that GhOPR9 might play an important role in cotton resistance to V. dahliae.

Molecular evidence for the involvement of cotton GhGLP2, in enhanced resistance to Verticillium and Fusarium Wilts and oxidative stress.

Germin-like proteins (GLPs) are a diverse and ubiquitous family of plant glycoproteins belonging to the cupin super family; they play considerable roles in plant responses against various abiotic and biotic stresses. Here, we provide evidence that GLP2 protein from cotton (Gossypium hirsutum) functions in plant defense responses against Verticillium dahliae, Fusarium oxysporum and oxidative stress. Purified recombinant GhGLP2 exhibits superoxide dismutase (SOD) activity and inhibits spore germination of pathogens. Virus-induced silencing of GhGLP2 in cotton results in increased susceptibility to pathogens, plants exhibited severe wilt on leaves, enhanced vascular browning and suppressed callose deposition. Transgenic Arabidopsis (Arabidopsis thaliana) plants overexpressing GhGLP2 showed significant resistance to V. dahliae and F. oxysporum, with reduced mycelia growth, increased callose deposition and cell wall lignification at infection sites on leaves. The enhanced tolerance of GhGLP2-transgenic Arabidopsis to oxidative stress was investigated by methyl viologen and ammonium persulfate treatments, along with increased H2O2 production. Further, the expression of several defense-related genes (PDF1.2, LOX2, and VSP1) or oxidative stress-related genes (RbohD, RbohF) was triggered by GhGLP2. Thus, our results confirmed the involvement of GhGLP2 in plant defense response against Verticillium and Fusarium wilt pathogens and stress conditions.

The ghr-miR164 and GhNAC100 modulate cotton plant resistance against Verticillium dahlia.

MicroRNAs (miRNAs) participate in plant development and defence through post-transcriptional regulation of the target genes. However, few miRNAs were reported to regulate cotton plant disease resistance. Here, we characterized the cotton miR164-NAC100 module in the later induction stage response of the plant to Verticillium dahliae infection. The results of GUS fusing reporter and transcript identity showed that ghr-miR164 can directly cleave the mRNA of GhNAC100 in the post-transcriptional process. The ghr-miR164 positively regulated the cotton plant resistance to V. dahliae according to analyses of its over-expression and knockdown. In link with results, the knockdown of GhNAC100 increased the plant resistance to V. dahliae. Based on LUC reporter, expression analyses and yeast one-hybrid (Y1H) assays, GhNAC100 bound to the CGTA-box of GhPR3 promoter and repressed its expression, negatively regulating plant disease resistance. These results showed that the ghr-miR164 and GhNAC100 module fine-tunes plant defence through the post-transcriptional regulation, which documented that miRNAs play important roles in plant resistance to vascular disease.

GbCYP86A1-1 from Gossypium barbadense positively regulates defence against Verticillium dahliae by cell wall modification and activation of immune pathways.

Suberin acts as stress-induced antipathogen barrier in the root cell wall. CYP86A1 encodes cytochrome P450 fatty acid ω-hydroxylase, which has been reported to be a key enzyme for suberin biosynthesis; however, its role in resistance to fungi and the mechanisms related to immune responses remain unknown. Here, we identified a disease resistance-related gene, GbCYP86A1-1, from Gossypium barbadense cv. Hai7124. There were three homologs of GbCYP86A1 in cotton, which are specifically expressed in roots and induced by Verticillium dahliae. Among them, GbCYP86A1-1 contributed the most significantly to resistance. Silencing of GbCYP86A1-1 in Hai7124 resulted in severely compromised resistance to V. dahliae, while heterologous overexpression of GbCYP86A1-1 in Arabidopsis improved tolerance. Tissue sections showed that the roots of GbCYP86A1-1 transgenic Arabidopsis had more suberin accumulation and significantly higher C16-C18 fatty acid content than control. Transcriptome analysis revealed that overexpression of GbCYP86A1-1 not only affected lipid biosynthesis in roots, but also activated the disease-resistant immune pathway; genes encoding the receptor-like kinases (RLKs), receptor-like proteins (RLPs), hormone-related transcription factors, and pathogenesis-related protein genes (PRs) were more highly expressed in the GbCYP86A1-1 transgenic line than control. Furthermore, we found that when comparing V. dahliae -inoculated and noninoculated plants, few differential genes related to disease immunity were detected in the GbCYP86A1-1 transgenic line; however, a large number of resistance genes were activated in the control. This study highlights the role of GbCYP86A1-1 in the defence against fungi and its underlying molecular immune mechanisms in this process.

Melatonin enhances cotton immunity to Verticillium wilt via manipulating lignin and gossypol biosynthesis.

Plants endure challenging environments in which they are constantly threatened by diverse pathogens. The soil-borne fungus Verticillium dahliae is a devastating pathogen affecting many plant species including cotton, in which it significantly reduces crop yield and fiber quality. Melatonin involvement in plant immunity to pathogens has been reported, but the mechanisms of melatonin-induced plant resistance are unclear. In this study, the role of melatonin in enhancing cotton resistance to V. dahliae was investigated. At the transcriptome level, exogenous melatonin increased the expression of genes in phenylpropanoid, mevalonate (MVA), and gossypol pathways after V. dahliae inoculation. As a result, lignin and gossypol, the products of these metabolic pathways, significantly increased. Silencing the serotonin N-acetyltransferase 1 (GhSNAT1) and caffeic acid O-methyltransferase (GhCOMT) melatonin biosynthesis genes compromised cotton resistance, with reduced lignin and gossypol levels after V. dahliae inoculation. Exogenous melatonin pre-treatment prior to V. dahliae inoculation restored the level of cotton resistance reduced by the above gene silencing effects. Melatonin levels were higher in resistant cotton cultivars than in susceptible cultivars after V. dahliae inoculation. The findings indicate that melatonin affects lignin and gossypol synthesis genes in phenylpropanoid, MVA, and gossypol pathways, thereby enhancing cotton resistance to V. dahliae.

Screening Cotton Genotypes for Reniform Nematode Resistance.

A rapid non-destructive reniform nematode (Rotylenchulus reniformis) screening protocol is needed for the development of resistant cotton (Gossypium hirsutum) varieties to improve nematode management. Most protocols involve extracting vermiform nematodes or eggs from the cotton root system or potting soil to determine population density or reproduction rate. These approaches are generally time-consuming with a small number of genotypes evaluated. An alternative approach is described here in which the root system is visually examined for nematode infection. The protocol involves inoculating cotton seedling 7 days after planting with vermiform nematodes and determining the number of females attached to the root system 28 days after inoculation. Data are expressed as the number of females per gram of fresh root weight to adjust for variation in root growth. The protocol provides an excellent method for evaluating host-plant resistance associated with the ability of the nematode to establish an infection site; however, resistance that hinders nematode reproduction is not assessed. As with other screening protocols, variation is commonly observed in nematode infection among individual genotypes within and between experiments. Data are presented to illustrate the range of variation observed using the protocol. To adjust for this variation, control genotypes are included in experiments. Nonetheless, the protocol provides a simple and rapid method to evaluate host-plant resistance. The protocol has been successfully used to identify resistant accessions from the G. arboreum germplasm collection and evaluate segregating populations of more than 300 individuals to determine the genetics of resistance. A vegetative propagation method for recovering plants for resistance breeding was also developed. After removal of the root system for nematode evaluation, the vegetative shoot is replanted to allow the development of a new root system. More than 95% of the shoots typically develop a new root system with plants reaching maturity.

Enhanced resistance to Verticillium dahliae mediated by an F-box protein GhACIF1 from Gossypium hirsutum.

Avr9/Cf-9-INDUCED F-BOX1 (ACIF1) was first identified during screening of Avr9/Cf-9-elicited genes in tobacco. Further analysis revealed that ACIF1 was required for hypersensitive responses triggered by various elicitors in tobacco and tomato, indicating that it may be involved in various disease resistance. Here, we cloned its cotton (Gossypium hirsutum) homolog GhACIF1, which encodes an F-box protein. We show that GhACIF1 interacts with the putative SKP1-like protein, named GhSKP1. Disease resistance assays show that GhACIF1 enhances resistance to Verticillium dahliae in Arabidopsis plants, while silencing of GhACIF1 confers sensitivity to V. dahliae in cotton. Further analysis show that PevD1 elicitor activates hypersensitive and acquired immune response mediated by GhACIF1. Collectively, these results indicate that GhACIF1 contributes to protection against V. dahliae infection.

Overexpression of the Key Virulence Factor 1,3-1,4-ß-d-Glucanase in the Endophytic Bacterium Bacillus halotolerans Y6 To Improve Verticillium Resistance in Cotton.

Verticillium wilt, caused by Verticillium dahliae, results in a dramatic loss of cotton yields in China. There is great potential for biocontrol to manage this destructive crop disease. In this study, we obtained the endophytic bacterium Bacillus halotolerans Y6 from Verticillium wilt-resistant cotton Gossypium barbadense Xinhai15; this bacterium possesses strong antagonistic abilities that inhibit V. dahliae spore germination and mycelial growth. The results of the enzyme activity assay, heterologous expression, and gene knockdown showed that the key virulence factor of Y6 for antagonizing V. dahliae was ß -glucanase Bgy6. To facilitate field tests of biological control, we constructed the homologous Bgy6-overexpression strain OY6. Compared with the wild-type Y6 strain, the ß-glucanase activity of OY6 was increased by 91.79%, and the inhibition rate of OY6 against V. dahliae V991 exceeded 96.7%. Moreover, the spores of V. dahliae V991 treated with OY6 showed more mucus and larger holes on the surface, as observed by scanning electron microscopy. Potting test results illustrated that both OY6 and Y6 could improve the resistance of upland cotton to Verticillium wilt. With the inoculation of V. dahliae V991 for 45 days, the disease index of G. hirsutum TM-1 treated with OY6 was only 8.33, which was significantly lower than that in plants treated with the wild-type strain Y6 (17.86) or the controls without bacteria (35.94). Our research provides a new idea for the control of Verticillium wilt in upland cotton via transforming endophytic bacteria of Verticillium wilt-resistant cotton and proposes a new solution to prevent and control Verticillium wilt.

RNA-Sequencing, Physiological and RNAi Analyses Provide Insights into the Response Mechanism of the ABC-Mediated Resistance to Verticillium dahliae Infection in Cotton.

Verticillium wilt that is caused by Verticillium dahliae, does result in massive annual yield losses and fiber quality decline in cotton. Control by conventional mechanisms is not possible due to a wide host range and the longevity of dormant fungi in the soil in the case of absence of a suitable host. Plants have developed various mechanisms to boost their immunity against various diseases, and one is through the induction of various genes. In this research, we carried out RNA sequencing and then identified the members of the adenosine triphosphate (ATP)-binding cassette (ABC) proteins to be critical in enhancing resistance to V. dahliae infection. A total of 166 proteins that are encoded by the ABC genes were identified in Gossypium raimondii with varying physiochemical properties. A novel ABC gene, Gorai.007G244600 (ABCF5), was found to be highly upregulated, and its homolog in the tetraploid cotton Gh_D11G3432 (ABCF5), was then silenced through virus induced gene silencing (VIGS) in G. hirsutum, tetraploid upland cotton. The mutant cotton seedlings ability to tolerate V. dahliae infection was significantly reduced. Based on the evaluation of oxidant enzymes, hydrogen peroxide (H2O2) and malondialdehyde (MDA) showed significantly increased levels in the leaves of the mutant compared to the wild type. In addition, antioxidant enzymes, peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) concentrations were reduced in the mutant cotton leaves after treatment with V. dahliae fungi as compared to the wild type. Moreover, expression levels of the biotic stress genes, cotton polyamine oxidase (GhPAO), cotton ribosomal protein L18 (GhRPL18), and cotton polygalacturonase-inhibiting protein-1 (GhPGIP1), were all downregulated in the mutant but they were highly upregulated in the various tissues of the wild cotton seedlings. This research has shown that ABC genes could play an important role in enhancing the immunity of cotton to V. dahliae infection, and thus can be explored in developing more resilient cotton genotypes with improved resistance to V. dahliae infection in cotton.