Cotton GhBAK1 Mediates Verticillium Wilt Resistance and Cell Death

Virus-induced gene silencing (VIGS) offers a powerful approach for functional analysis of individual genes by knocking down their expression. We have adopted this approach to dissect gene functions in cotton resistant to Verticillium wilt, one of the most devastating diseases worldwide. We showed here that highly efficient VIGS was obtained in a cotton breeding line (CA4002) with partial resistance to Verticillium wilt, and GhMKK2 and GhVe1 are required for its resistance to Verticillium wilt. Arabidopsis AtBAK1/SERK3, a central regulator in plant disease resistance, belongs to a subfamily of somatic embryogenesis receptor kinases (SERKs) with five members, AtSERK1 to AtSERK5. Two BAK1 orthologs and one SERK1 ortholog were identified in the cotton genome. Importantly, GhBAK1 is required for CA4002 resistance to Verticillium wilt. Surprisingly, silencing of GhBAK1 is sufficient to trigger cell death accompanied with production of reactive oxygen species in cotton. This result is distinct from Arabidopsis in which AtBAK1 and AtSERK4 play redundant functions in cell death control. Apparently, cotton has only evolved SERK1 and BAK1 whereas AtSERK4/5 are newly evolved genes in Arabidopsis. Our studies indicate the functional importance of BAK1 in Verticillium wilt resistance and suggest the dynamic evolution of SERK family members in different plant species.     

The Gossypium hirsutum TIR-NBS-LRR gene GhDSC1 mediates resistance against Verticillium wilt

Improving genetic resistance is a preferred method to manage Verticillium wilt of cotton and other hosts. Identifying host resistance is difficult because of the dearth of resistance genes against this pathogen. Previously, a novel candidate gene involved in Verticillium wilt resistance was identified by a genome-wide association study using a panel of Gossypium hirsutum accessions. In this study, we cloned the candidate resistance gene from cotton that encodes a protein sharing homology with the TIR-NBS-LRR receptor-like defence protein DSC1 in Arabidopsis thaliana (hereafter named GhDSC1). GhDSC1 expressed at higher levels in response to Verticillium wilt and jasmonic acid (JA) treatment in resistant cotton cultivars as compared to susceptible cultivars and its product was localized to nucleus. The transfer of GhDSC1 to Arabidopsis conferred Verticillium resistance in an A. thaliana dsc1 mutant. This resistance response was associated with reactive oxygen species (ROS) accumulation and increased expression of JA-signalling-related genes. Furthermore, the expression of GhDSC1 in response to Verticillium wilt and JA signalling in A. thaliana displayed expression patterns similar to GhCAMTA3 in cotton under identical conditions, suggesting a coordinated DSC1 and CAMTA3 response in A. thaliana to Verticillium wilt. Analyses of GhDSC1 sequence polymorphism revealed a single nucleotide polymorphism (SNP) difference between resistant and susceptible cotton accessions, within the P-loop motif encoded by GhDSC1. This SNP difference causes ineffective activation of defence response in susceptible cultivars. These results demonstrated that GhDSC1 confers Verticillium resistance in the model plant system of A. thaliana, and therefore represents a suitable candidate for the genetic engineering of Verticillium wilt resistance in cotton.     

基因沉默技术在抗真菌病害中的应用和展望

真菌病害严重威胁作物的产量和品质,给国家和人民造成巨大的经济损失。尤其是引起维管束病害的土传真菌,化学农药的作用效果很不理想。利用抗性基因进行遗传育种是目前生物防治的重要手段之一,但对于缺乏抗性资源的物种,面对强大的土壤真菌病害,研究者也时常束手无策。近年来,利用RNA干扰技术发展而来的宿主诱导的基因沉默(Host induced gene silencing,HIGS)策略,在抗病虫害领域逐渐崭露头角,但由于真菌侵染的复杂多样性及土壤传播的特性,HIGS在土壤真菌病害中的应用充满神秘和挑战。本研究室近期揭示了棉花黄萎病(一种严重的土壤真菌病害)的"罪魁祸首"——大丽轮枝菌的侵染结构和侵染过程;并首次证明了宿主植物内源小RNA能够跨界进入病原菌细胞中降解致病基因表达的抗病作用;在此基础上,本研究室利用HIGS在棉花上获得了对黄萎病抗性较高的品系,成功地开辟了抗土壤黄萎真菌病害的新天地,研究结果显示出基因沉默技术在这一领域强大的应用潜力和前景。     

Scaffold protein GhMORG1 enhances the resistance of cotton to Fusarium oxysporum by facilitating the MKK6‐MPK4 cascade

In eukaryotes, MAPK scaffold proteins are crucial for regulating the function of MAPK cascades. However, only a few MAPK scaffold proteins have been reported in plants, and the molecular mechanism through which scaffold proteins regulate the function of the MAPK cascade remains poorly understood. Here, we identified GhMORG1, a GhMKK6‐GhMPK4 cascade scaffold protein that positively regulates the resistance of cotton to Fusarium oxysporum. GhMORG1 interacted with GhMKK6 and GhMPK4, and the overexpression of GhMORG1 in cotton protoplasts dramatically increased the activity of the GhMKK6‐GhMPK4 cascade. Quantitative phosphoproteomics was used to clarify the mechanism of GhMORG1 in regulating disease resistance, and thirty‐two proteins were considered as the putative substrates of the GhMORG1‐dependent GhMKK6‐GhMPK4 cascade. These putative substrates were involved in multiple disease resistance processes, such as cellular amino acid metabolic processes, calcium ion binding and RNA binding. The kinase assays verified that most of the putative substrates were phosphorylated by the GhMKK6‐GhMPK4 cascade. For functional analysis, nine putative substrates were silenced in cotton, respectively. The resistance of cotton to F. oxysporum was decreased in the substrate‐silenced cottons. These results suggest that GhMORG1 regulates several different disease resistance processes by facilitating the phosphorylation of GhMKK6‐GhMPK4 cascade substrates. Taken together, these findings reveal a new plant MAPK scaffold protein and provide insights into the mechanism of plant resistance to pathogens.     

Cotton GhMKK5 affects disease resistance, induces HR-like cell death, and reduces the tolerance to salt and drought stress in transgenic Nicotiana benthamiana

Mitogen-activated protein kinase (MAPK) cascades are involved in various processes from plant growth and development to biotic and abiotic stress responses. MAPK kinases (MAPKKs), which link MAPKs and MAPKK kinases (MAPKKKs), play crucial roles in MAPK cascades to mediate a variety of stress responses in plants. However, few MAPKKs have been functionally characterized in cotton (Gossypium hirsutum). In this study, a novel gene, GhMKK5, from cotton belonging to the group C MAPKKs was isolated and characterized. The expression of GhMKK5 can be induced by pathogen infection, abiotic stresses, and multiple defence-related signal molecules. The overexpression of GhMKK5 in Nicotiana benthamiana enhanced the plants' resistance to the bacterial pathogen Ralstonia solanacearum by elevating the expression of pathogen resistance (PR) genes, including PR1a, PR2, PR4, PR5, and NPR1, but increased the plants' sensitivity to the oomycete pathogen Phytophthora parasitica var. nicotianae Tucker. Importantly, GhMKK5-overexpressing plants displayed markedly elevated expression of reactive oxygen species-related and cell death marker genes, such as NtRbohA and NtCDM, and resulted in hypersensitive response (HR)-like cell death characterized by the accumulation of H(2)O(2). Furthermore, it was demonstrated that GhMKK5 overexpression in plants reduced their tolerance to salt and drought stresses, as determined by statistical analysis of seed germination, root length, leaf water loss, and survival rate. Drought obviously accelerated the cell death phenomenon in GhMKK5-overexpressing plants. These results suggest that GhMKK5 may play an important role in pathogen infection and the regulation of the salt and drought stress responses in plants.     

Quantitative trait locus analysis of Verticillium wilt resistance in an introgressed recombinant inbred population of Upland cotton

Verticillium wilt (VW) of Upland cotton (Gossypium hirsutum L.) is caused by the soil-borne fungal pathogen Verticillium dahlia Kleb. The availability of VW-resistant cultivars is vital for control of this economically important disease, but there is a paucity of Upland cotton breeding lines and cultivars with a high level of resistance to VW. In general, G. barbadense L. (source of Pima cotton) is more VW-resistant than Upland cotton. However, the transfer of VW resistance from G. barbadense to Upland cotton is challenging because of hybrid breakdown in the F2 and successive generations of interspecific populations. We conducted two replicated greenhouse studies (tests 1 and 2) to assess the heritability of VW resistance to a defoliating V. dahliae isolate and identify genetic markers associated with VW resistance in an Upland cotton recombinant inbred mapping population that has stable introgression from Pima cotton. Disease ratings at the seedling stage on several different days after the first inoculation (DAI) in test 1, as well as the percentages of infected and defoliated leaves at 2 DAI in test 2, were found to be low to moderately heritable, indicating the importance of a replicated progeny test in selection for VW resistance. With a newly constructed linkage map consisting of 882 simple sequence repeat, single nucleotide polymorphism, and resistance gene analog–amplified fragment length polymorphism marker loci, we identified a total of 21 quantitative trait loci (QTLs) on 11 chromosomes and two linkage groups associated with VW resistance at several different DAIs in greenhouse tests 1 and 2. The markers associated with the VW resistance QTLs will facilitate fine mapping and cloning of VW resistance genes and genomics-assisted breeding for VW-resistant cultivars.     

Interfamily transfer of tomato Ve1 mediates Verticillium resistance in Arabidopsis

Vascular wilts caused by soil-borne fungal species of the Verticillium genus are devastating plant diseases. The most common species, Verticillium dahliae and Verticillium albo-atrum, have broad host ranges and are notoriously difficult to control. Therefore, genetic resistance is the preferred method for disease control. Only from tomato (Solanum lycopersicum) has a Verticillium resistance locus been cloned, comprising the Ve1 gene that encodes a receptor-like protein-type cell surface receptor. Due to lack of a suitable model for receptor-like protein (RLP)-mediated resistance signaling in Arabidopsis (Arabidopsis thaliana), so far relatively little is known about RLP signaling in pathogen resistance. Here, we show that Ve1 remains fully functional after interfamily transfer to Arabidopsis and that Ve1-transgenic Arabidopsis is resistant to race 1 but not to race 2 strains of V. dahliae and V. albo-atrum, nor to the Brassicaceae-specific pathogen Verticillium longisporum. Furthermore, we show that signaling components utilized by Ve1 in Arabidopsis to establish Verticillium resistance overlap with those required in tomato and include SERK3/BAK1, EDS1, and NDR1, which strongly suggests that critical components for resistance signaling are conserved. We subsequently investigated the requirement of SERK family members for Ve1 resistance in Arabidopsis, revealing that SERK1 is required in addition to SERK3/BAK1. Using virus-induced gene silencing, the requirement of SERK1 for Ve1-mediated resistance was confirmed in tomato. Moreover, we show the requirement of SERK1 for resistance against the foliar fungal pathogen Cladosporium fulvum mediated by the RLP Cf-4. Our results demonstrate that Arabidopsis can be used as model to unravel the genetics of Ve1-mediated resistance.     

Genome‐wide association study discovered candidate genes of Verticillium wilt resistance in upland cotton (Gossypium hirsutum L.)

Verticillium wilt (VW), caused by infection by Verticillium dahliae, is considered one of the most yield‐limiting diseases in cotton. To examine the genetic architecture of cotton VW resistance, we performed a genome‐wide association study (GWAS) using a panel of 299 accessions and 85 630 single nucleotide polymorphisms (SNPs) detected using the specific‐locus amplified fragment sequencing (SLAF‐seq) approach. Trait–SNP association analysis detected a total of 17 significant SNPs at P < 1.17 × 10^–5 (P = 1/85 630, –log₁₀P = 4.93); the peaks of SNPs associated with VW resistance on A10 were continuous and common in three environments (RDIG2015, RDIF2015 and RDIF2016). Haplotype block structure analysis predicted 22 candidate genes for VW resistance based on A10_99672586 with a minimum P‐value (–log₁₀P = 6.21). One of these genes (CG02) was near the significant SNP A10_99672586 (0.26 Mb), located in a 372‐kb haplotype block, and its Arabidopsis AT3G25510 homologues contain TIR‐NBS‐LRR domains that may be involved in disease resistance response. Real‐time quantitative PCR and virus‐induced gene silencing (VIGS) analysis showed that CG02 was specific to up‐regulation in the resistant (R) genotype Zhongzhimian2 (ZZM2) and that silenced plants were more susceptible to V. dahliae. These results indicate that CG02 is likely the candidate gene for resistance against V. dahliae in cotton. The identified locus or gene may serve as a promising target for genetic engineering and selection for improving resistance to VW in cotton.     

The cotton MAPK kinase GhMPK20 negatively regulates resistance to Fusarium oxysporum by mediating the MKK4–MPK20–WRKY40 cascade

Fusarium wilt is one of the most serious diseases affecting cotton. However, the pathogenesis and mechanism by which Fusarium oxysporum overcomes plant defence responses are unclear. Here, a new group D mitogen‐activated protein kinase (MAPK) gene, GhMPK20, was identified and functionally analysed in cotton. GhMPK20 expression was significantly induced by F. oxysporum. Virus‐induced gene silencing (VIGS) of GhMPK20 in cotton increased the tolerance to F. oxysporum, whereas ectopic GhMPK20 overexpression in Nicotiana benthamiana reduced F. oxysporum resistance via disruption of the salicylic acid (SA)‐mediated defence pathway. More importantly, an F. oxysporum‐induced MAPK cascade pathway composed of GhMKK4, GhMPK20 and GhWRKY40 was identified. VIGS of GhMKK4 and GhWRKY40 also enhanced F. oxysporum resistance in cotton, and the function of GhMKK4–GhMPK20 was shown to be essential for F. oxysporum‐induced GhWRKY40 expression. Together, our results indicate that the GhMKK4–GhMPK20–GhWRKY40 cascade in cotton plays an important role in the pathogenesis of F. oxysporum. This research broadens our knowledge of the negative role of the MAPK cascade in disease resistance in cotton and provides an important scientific basis for the formulation of Fusarium wilt prevention strategies.     

陆地棉品种和骨干品系黄萎病抗性鉴定

选育和推广抗病品种是防治陆地棉黄萎病的主要措施,为了早日实现多类型、多区域大面积抗病品种的应用,本研究选取107份遗传背景差异较大的种质,利用河北省农林科学院棉花研究所小安舍试验站黄萎病病圃进行了3年黄萎病抗性重复鉴定。鉴定得到抗病品系8个,占7.5%;耐病品种(系)20个,占18.7%。本研究表明,当前被作为育种亲本的抗病品系还太少,需要深入开展抗病遗传机制,以及与其他经济性状协同改良的关系,为陆地棉抗病育种提供理论指导;达到抗病或接近抗病水平的大部分品种(系)来自于海陆野远缘后代,具有外源基因血统,证明了远缘杂交是陆地棉黄萎病抗性改良的有效手段。     

Island Cotton Gbve1 Gene Encoding A Receptor-Like Protein Confers Resistance to Both Defoliating and Non-Defoliating Isolates of Verticillium dahliae

Verticillium wilt caused by soilborne fungus Verticillium dahliae could significantly reduce cotton yield. Here, we cloned a tomato Ve homologous gene, Gbve1, from an island cotton cultivar that is resistant to Verticillium wilt. We found that the Gbve1 gene was induced by V. dahliae and by phytohormones salicylic acid, jasmonic acid, and ethylene, but not by abscisic acid. The induction of Gbve1 in resistant cotton was quicker and stronger than in Verticillium-susceptible upland cotton following V. dahliae inoculation. Gbve1 promoter-driving GUS activity was found exclusively in the vascular bundles of roots and stems of transgenic Arabidopsis. Virus-induced silencing of endogenous genes in resistant cotton via targeting a fragment of the Gbve1 gene compromised cotton resistance to V. dahliae. Furthermore, we transformed the Gbve1 gene into Arabidopsis and upland cotton through Agrobacterium-mediated transformation. Overexpression of the Gbve1 gene endowed transgenic Arabidopsis and upland cotton with resistance to high aggressive defoliating and non-defoliating isolates of V. dahliae. And HR-mimic cell death was observed in the transgenic Arabidopsis. Our results demonstrate that the Gbve1 gene is responsible for resistance to V. dahliae in island cotton and can be used for breeding cotton varieties that are resistant to Verticillium wilt.     

Molecular mapping of Verticillium wilt resistance QTL clustered on chromosomes D7 and D9 in upland cotton

Verticillium wilt is a destructive disease with international consequences for cotton production. Breeding broad-spectrum resistant cultivars is considered to be one of the most effective means for reducing crop losses. A resistant cotton cultivar, 60182, was crossed with a susceptible cultivar, Jun-mian 1, to identify markers for Verticillium resistance genes and validate the mode of its inheritance. Genetic segregation analysis for Verticillium wilt resistance was evaluated based upon infected leaf percentage in the seedling stage using major gene-polygene mixed inheritance models and joint analysis of P₁, P₂, F₁, B₁, B₂ and F₂ populations obtained from the cultivar cross. We found that resis-tance of upland cotton cultivar 60182 to isolates BP2, VD8 and T9, and their isoconcentration mixture was controlled by two major genes with additive-dominance-epistatic effects, and the inheritance of the major gene was dominant. Furthermore, a genetic linkage map was constructed using F₂ segregating population and resistance phenotypic data were obtained using F_2︰3 families inoculated with different isolates and detected in different developmental stages. The genetic linkage map with 139 loci was comprised of 31 linkage groups covering 1165 cM, with an average distance of 8.38 cM between two markers, or 25.89% of the cotton genome length. From 60182, we found 4 QTL on chromosome D7 and 4 QTL on D9 for BP2, 5 QTL on D7 and 9 QTL on D9 for VD8, 4 QTL on D7 and 5 QTL on D9 for T9 and 3 QTL on D7 and 7 QTL on D7 for mixed pathogens. The QTL mapping results revealed that QTL clusters with high contribution rates were screened simultaneously on chromosomes D9 and D7 by multiple interval mapping (CIM), whether from resistance phenotypic data from different developmental stages or for different isolates. The result is consistent with the genetic model of two major genes in 60182 and suggests broad-spectrum resistance to both defoliating isolates of V. dahliae and nondefoliating iso-lates. The markers associated with resistance QTL may facilitate the use of Verticillium wilt resistance genes in improving breeding programs for cotton.     

Identification of two blackleg resistance genes and fine mapping of one of these two genes in a <Emphasis Type="Italic">Brassica napus</Emphasis> canola cultivar ‘Surpass 400’

Blackleg resistant cultivars have been developed through conventional breeding methods and are successfully used globally to control this disease in canola production. To clone blackleg resistance genes and to understand the mechanism underlying the resistance, a blackleg resistant canola cultivar ‘Surpass 400’ was used to develop a gene mapping population. A previously reported high density genetic map was used to find a resistance gene region that corresponded to linkage group N10 in B. napus. Differential interactions between the resistant lines and a pathogen isolate were discovered with two resistance genes BLMR1 and BLMR2 identified through linkage analysis of five genome-specific molecular markers. BLMR1 provides resistance through the hypersensitive response that protects inoculated cotyledons from becoming infected, Unlike BLMR1, BLMR2 slows down the development of individual infection loci. BLMR1 and BLMR2 segregated independently in two large F₃BC₂ populations. Fine mapping of BLMR1 was performed with 12 genome-specific molecular markers. The closest marker with a genetic distance of 0.13 cM to BLMR1 was identified, which lays a solid foundation for cloning BLMR1.     

The Arabidopsis thaliana DNA-binding protein AHL19 mediates verticillium wilt resistance

Verticillium spp. are destructive soilborne fungal pathogens that cause vascular wilt diseases in a wide range of plant species. Verticillium wilts are particularly notorious, and genetic resistance in crop plants is the most favorable means of disease control. In a gain-of-function screen using an activation-tagged Arabidopsis mutant collection, we identified four mutants, A1 to A4, which displayed enhanced resistance toward the vascular wilt species Verticillium dahliae, V. albo-atrum and V. longisporum but not to Fusarium oxysporum f. sp. raphani. Further testing revealed that mutant A2 displayed enhanced Ralstonia solanacearum resistance, while mutants A1 and A3 were more susceptible toward Pseudomonas syringae pv. tomato. Identification of the activation tag insertion site in the A1 mutant revealed an insertion in close proximity to the gene encoding AHL19, which was constitutively expressed in the mutant. AHL19 knock-out alleles were found to display enhanced Verticillium susceptibility whereas overexpression of AHL19 resulted in enhanced Verticillium resistance, showing that AHL19 acts as a positive regulator of plant defense.