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.     

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。     

The island cotton NBS‐LRR gene GbaNA1 confers resistance to the non‐race 1 Verticillium dahliae isolate Vd991

Wilt caused by Verticillium dahliae significantly reduces cotton yields, as host resistance in commercially cultivated Gossypium species is lacking. Understanding the molecular basis of disease resistance in non‐commercial Gossypium species could galvanize the development of Verticillium wilt resistance in cultivated species. Nucleotide‐binding site leucine‐rich repeat (NBS‐LRR) proteins play a central role in plant defence against pathogens. In this study, we focused on the relationship between a locus enriched with eight NBS‐LRR genes and Verticillium wilt resistance in G. barbadense. Independent virus‐induced gene silencing of each of the eight NBS‐LRR genes in G. barbadense cultivar Hai 7124 revealed that silencing of GbaNA1 alone compromised the resistance of G. barbadense to V. dahliae isolate Vd991. In cultivar Hai 7124, GbaNA1 could be induced by V. dahliae isolate Vd991 and by ethylene, jasmonic acid and salicylic acid. Nuclear protein localization of GbaNA1 was demonstrated by transient expression. Sequencing of the GbaNA1 orthologue in nine G. hirsutum accessions revealed that all carried a non‐functional allele, caused by a premature peptide truncation. In addition, all 10 G. barbadense and nine G. hirsutum accessions tested carried a full‐length (～1140 amino acids) homologue of the V. dahliae race 1 resistance gene Gbve1, although some sequence polymorphisms were observed. Verticillium dahliae Vd991 is a non‐race 1 isolate that lacks the Ave1 gene. Thus, the resistance imparted by GbaNA1 appears to be mediated by a mechanism distinct from recognition of the fungal effector Ave1.     

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.     

The cell biology of lignification in higher plants

Background Lignin is a polyphenolic polymer that strengthens and waterproofs the cell wall of specialized plant cell types. Lignification is part of the normal differentiation programme and functioning of specific cell types, but can also be triggered as a response to various biotic and abiotic stresses in cells that would not otherwise be lignifying.Scope Cell wall lignification exhibits specific characteristics depending on the cell type being considered. These characteristics include the timing of lignification during cell differentiation, the palette of associated enzymes and substrates, the sub-cellular deposition sites, the monomeric composition and the cellular autonomy for lignin monomer production. This review provides an overview of the current understanding of lignin biosynthesis and polymerization at the cell biology level.Conclusions The lignification process ranges from full autonomy to complete co-operation depending on the cell type. The different roles of lignin for the function of each specific plant cell type are clearly illustrated by the multiple phenotypic defects exhibited by knock-out mutants in lignin synthesis, which may explain why no general mechanism for lignification has yet been defined. The range of phenotypic effects observed include altered xylem sap transport, loss of mechanical support, reduced seed protection and dispersion, and/or increased pest and disease susceptibility.     

Genetic analysis of Verticillium wilt resistance in a backcross inbred line population and a meta-analysis of quantitative trait loci for disease resistance in cotton

Background

Verticillium wilt (VW) and Fusarium wilt (FW), caused by the soil-borne fungi Verticillium dahliae and Fusarium oxysporum f. sp. vasinfectum, respectively, are two most destructive diseases in cotton production worldwide. Root-knot nematodes (Meloidogyne incognita, RKN) and reniform nematodes (Rotylenchulus reniformis, RN) cause the highest yield loss in the U.S. Planting disease resistant cultivars is the most cost effective control method. Numerous studies have reported mapping of quantitative trait loci (QTLs) for disease resistance in cotton; however, very few reliable QTLs were identified for use in genomic research and breeding.

Results

This study first performed a 4-year replicated test of a backcross inbred line (BIL) population for VW resistance, and 10 resistance QTLs were mapped based on a 2895 cM linkage map with 392 SSR markers. The 10 VW QTLs were then placed to a consensus linkage map with other 182 VW QTLs, 75 RKN QTLs, 27 FW QTLs, and 7 RN QTLs reported from 32 publications. A meta-analysis of QTLs identified 28 QTL clusters including 13, 8 and 3 QTL hotspots for resistance to VW, RKN and FW, respectively. The number of QTLs and QTL clusters on chromosomes especially in the A-subgenome was significantly correlated with the number of nucleotide-binding site (NBS) genes, and the distribution of QTLs between homeologous A- and D- subgenome chromosomes was also significantly correlated.

Conclusions

Ten VW resistance QTL identified in a 4-year replicated study have added useful information to the understanding of the genetic basis of VW resistance in cotton. Twenty-eight disease resistance QTL clusters and 24 hotspots identified from a total of 306 QTLs and linked SSR markers provide important information for marker-assisted selection and high resolution mapping of resistance QTLs and genes. The non-overlapping of most resistance QTL hotspots for different diseases indicates that their resistances are controlled by different genes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1682-2) contains supplementary material, which is available to authorized users.     

Genetic analysis of Verticillium wilt tolerance in cotton using pedigree data from three crosses

Summary Three crosses and descendant generations were used in a field study of the inheritance of tolerance to Verticillium wilt, caused by Verticillium dahliae Kleb., in upland cotton (Gossypium hirsutum L.). The tolerant cultivar Acala SJC-1 was crossed to more susceptible parents, breeding line S5971 and cultivars Acala 4-42 and Deltapine 70. Seven generations were evaluated for each cross: the two parents (P₁ and P₂), F₁; F₂, F₃, and reciprocal backcrosses (B₁ and B₂). The genetic control of tolerance in these crosses appears to involve more than one gene, based on an unsatisfactory fit to expected phenotypic distributions for the generations under a single-locus model. An analysis of generation means indicated that pooled additive and pooled dominance effects over loci were adequate to explain the variation among generations for crosses of SJC-1 X S5971 and SJC-1 X DPL70. Tolerance in these crosses appeared to be controlled by recessive factors. For the SJC-1 X 4-42 cross, an adequate fit to a digenic epistatic model was not possible, and none of the genetic parameters except the F₂ mean were significant. Heritabilities for tolerance to Verticillium wilt, determined from regressions of F₃ progeny on F₂ parents for the crosses of SJC-1 X S5971 and SJC-1 X DPL70, ranged from 0.12 to 0.28. Therefore, individual plant selection for improved tolerance is expected to be inefficient.Contribution from the Department of Botany and Plant Sciences, University of California, Riverside, CA 92521, USA, to be included in a dissertation by the senior author in partial fulfillment of the Ph. D. degree     

海岛棉GbHyPRP1克隆及其转基因拟南芥抗黄萎病验证

在对黄萎病菌胁迫处理的海岛棉Pima 90-53根组织全长c DNA文库分析中,筛选到一个与黄萎病胁迫相关的杂合富含脯氨酸蛋白(hybrid proline-rich protein)基因,将其命名为Gb Hy PRP1。该基因c DNA序列全长1747 bp,开放阅读框945 bp,编码一个由314个氨基酸残基组成的蛋白,包含信号肽、N端富含脯氨酸域及C端Pollen Ole e I域。同源序列分析显示,Gb Hy PRP1与来自雷蒙德氏棉、陆地棉和亚洲棉的Hy PRP1蛋白序列相似性最高,分别为95.95%、93.87%和91.34%。q RT-PCR分析结果显示,受黄萎病菌胁迫后海岛棉根部Gb Hy PRP1表达显著下调。将Gb Hy PRP1基因克隆至植物超表达载体,农杆菌介导转化拟南芥获得转基因植株。病指统计分析表明Gb Hy PRP1过量表达显著降低了拟南芥对黄萎病的抗性。据此推测Gb Hy PRP1参与棉花抗黄萎病,可能是一个重要的负调控因子。     

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.     

Changes in the promoter of a defender against apoptotic cell death gene affect its expression in upland cotton

The DAD1 (defender against apoptotic cell death) gene is a negative regulator of programmed cell death. It plays important roles in plant growth, development, environmental adaptation, and aging. We examined whether and how the expression of DAD1 gene is affected by the promoter changes in the allotetraploid upland cotton (Gossypium hirsutum L.). We compared the expression status of the genes in the leaves of an elite cultivar and a wild variety of G. hirsutum, and cloned and analyzed a 1.3-kb promoter region of the genes in two cotton types. Results revealed that the expression of a subgenomic homoeolog of DAD1 (DAD1 _At ) was significantly upregulated in leaves of the cultivated cotton when compared with that in a wild cotton. In addition, we identified 18 variations, including 14 single nucleotide polymorphisms and four indels between the promoters of the two cotton types. We detected at least 10 of 18 variations in the promoters related to possible cis-regulatory elements. Further transient tobacco assays using heterogeneous DAD1 _At promoter::GUS fusion showed that the activity of the DAD1 _At promoter in the cultivated cotton was stronger than that in the wild cotton, which suggested a dynamic change in the DAD1 _At promoter during genetic divergence of the two cottons. This study provides an example of how gene expression could be affected by specific changes in the gene promoter.     

Transfer of tomato immune receptor Ve1 confers Ave1‐dependent Verticillium resistance in tobacco and cotton

Verticillium wilts caused by soilborne fungal species of the Verticillium genus are economically important plant diseases that affect a wide range of host plants and are notoriously difficult to combat. Perception of pathogen(‐induced) ligands by plant immune receptors is a key component of plant innate immunity. In tomato, race‐specific resistance to Verticillium wilt is governed by the cell surface‐localized immune receptor Ve1 through recognition of the effector protein Ave1 that is secreted by race 1 strains of Verticillium spp. It was previously demonstrated that transgenic expression of tomato Ve1 in the model plant Arabidopsis thaliana leads to Verticillium wilt resistance. Here, we investigated whether tomato Ve1 can confer Verticillium resistance when expressed in the crop species tobacco (Nicotiana tabcum) and cotton (Gossypium hirsutum). We show that transgenic tobacco and cotton plants constitutively expressing tomato Ve1 exhibit enhanced resistance against Verticillium wilt in an Ave1‐dependent manner. Thus, we demonstrate that the functionality of tomato Ve1 in Verticillium wilt resistance through recognition of the Verticillium effector Ave1 is retained after transfer to tobacco and cotton, implying that the Ve1‐mediated immune signalling pathway is evolutionary conserved across these plant species. Moreover, our results suggest that transfer of tomato Ve1 across sexually incompatible plant species can be exploited in breeding programmes to engineer Verticillium wilt resistance.     

Vitrification of carnation in vitro: Changes in cell wall mechanical properties,cellulose and lignin content

Vitrification of internodes of carnation was brought about by culturing in liquid medium. Cell wall extensibility of these internodes was kinetically followed in comparison to that of normal plants using the constant stress method. Liquid culture induced increased immediate and total deformation capacities of the walls from the second day. Measurements indicated that these deformation capacities involved plastic properties rather than elastic ones. These changes were paralleled by decreased relative levels of cellulose and lignin.     

Ultrastructural changes and location of beta-1, 3-glucanase in resistant and susceptible cotton callus cells in response to treatment with toxin of Verticillium dahliae and salicylic acid

Calli from two cotton cultivars susceptible and resistant to Verticillium wilt, were treated with a crude toxin of Verticillium dahliae (VD-toxin) plus salicylic acid (SA). Cells treated with VD-toxin showed distinct ultrastructural changes. Cells from the susceptible cultivar displayed damage to plasma membrane and cytoplasm. The deleterious effect on cells of the resistant cultivar, with an accumulation of electron-dense precipitate in the vacuoles, was less noticeable. Exogenous SA protected callus cells from VD-toxin. We also report the localization of β-1,3-glucanase in callus cells with immunofluorescence labeling. Stronger fluorescence was observed in the extra-cellular space in resistant than in susceptible cotton; strongest in resistant cotton after 5 days of treatment with VD-toxin plus SA. The findings reported here indicate an important role of exogenous salicylic acid in the induction of resistance to VD-toxin in cotton. Coupled with an increase in β-1,3-glucanase, cellular integrity is maintained and damage to cell wall and plasma membrane is avoided.     

Ectopic expression of a novel OsExtensin‐like gene consistently enhances plant lodging resistance by regulating cell elongation and cell wall thickening in rice

Plant lodging resistance is an important integrative agronomic trait of grain yield and quality in crops. Although extensin proteins are tightly associated with plant cell growth and cell wall construction, little has yet been reported about their impacts on plant lodging resistance. In this study, we isolated a novel extensin‐like (OsEXTL) gene in rice, and selected transgenic rice plants that expressed OsEXTL under driven with two distinct promoters. Despite different OsEXTL expression levels, two‐promoter‐driven OsEXTL‐transgenic plants, compared to a rice cultivar and an empty vector, exhibited significantly reduced cell elongation in stem internodes, leading to relatively shorter plant heights by 7%–10%. Meanwhile, the OsEXTL‐transgenic plants showed remarkably thickened secondary cell walls with higher cellulose levels in the mature plants, resulting in significantly increased detectable mechanical strength (extension and pushing forces) in the mature transgenic plants. Due to reduced plant height and increased plant mechanical strength, the OsEXTL‐transgenic plants were detected with largely enhanced lodging resistances in 3 years field experiments, compared to those of the rice cultivar ZH11. In addition, despite relatively short plant heights, the OsEXTL‐transgenic plants maintain normal grain yields and biomass production, owing to their increased cellulose levels and thickened cell walls. Hence, this study demonstrates a largely improved lodging resistance in the OsEXTL‐transgenic rice plants, and provides insights into novel extensin functions in plant cell growth and development, cell wall network construction and wall structural remodelling.     

Expression of the Talaromyces flavus glucose oxidase gene in cotton and tobacco reduces fungal infection,but is also phytotoxic

Glucose oxidase secreted by the fungus Talaromyces flavus generates, in the presence of glucose, hydrogen peroxide that is toxic to phytopathogenic fungi responsible for economically important diseases in many crops. A glucose oxidase gene from T. flavus, was modified with a carrot extensin signal peptide and fused to either a constitutive or root-specific plant promoter. T1 tobacco plants expressing the enzyme constitutively were protected against infection by the seedling pathogen Rhizoctonia solani. Constitutive expression in tobacco was associated with reduced root growth, and slow germination on culture medium, and with reduced seed set in glasshouse conditions. Several independent transformed cotton plants with a root-specific construct expressed high glucose oxidase activity in the roots, excluding the root tip. Selected T3 homozygous lines showed some protection against the root pathogen, Verticillium dahliae, but not against Fusarium oxysporum. High levels of glucose oxidase expression in cotton roots were associated with reduced height, seed set and seedling germination and reduced lateral root formation. If this gene is to be of value for crop protection against pathogens it will require precise control of its expression to remove the deleterious phenotypes. This revised version was published online in June 2006 with corrections to the Cover Date.