Cotton 4-coumarate-CoA ligase 3 enhanced plant resistance to Verticillium dahliae by promoting jasmonic acid signaling-mediated vascular lignification and metabolic flux.

Lignins and their antimicrobial-related polymers cooperatively enhance plant resistance to pathogens. Several isoforms of 4-coumarate-coenzyme A ligases (4CLs) have been identified as indispensable enzymes involved in lignin and flavonoid biosynthetic pathways. However, their roles in plant-pathogen interaction are still poorly understood. This study uncovers the role of Gh4CL3 in cotton resistance to the vascular pathogen Verticillium dahliae. The cotton 4CL3-CRISPR/Cas9 mutant (CR4cl) exhibited high susceptibility to V. dahliae. This susceptibility was most probably due to the reduction in the total lignin content and the biosynthesis of several phenolic metabolites, e.g., rutin, catechin, scopoletin glucoside, and chlorogenic acid, along with jasmonic acid (JA) attenuation. These changes were coupled with a significant reduction in 4CL activity toward p-coumaric acid substrate, and it is likely that recombinant Gh4CL3 could specifically catalyze p-coumaric acid to form p-coumaroyl-coenzyme A. Thus, overexpression of Gh4CL3 (OE4CL) showed increasing 4CL activity that augmented phenolic precursors, cinnamic, p-coumaric, and sinapic acids, channeling into lignin and flavonoid biosyntheses and enhanced resistance to V. dahliae. Besides, Gh4CL3 overexpression activated JA signaling that instantly stimulated lignin deposition and metabolic flux in response to pathogen, which all established an efficient plant defense response system, and inhibited V. dahliae mycelium growth. Our results propose that Gh4CL3 acts as a positive regulator for cotton resistance against V. dahliae by promoting JA signaling-mediated enhanced cell wall rigidity and metabolic flux.

The elicitor VP2 from Verticillium dahliae triggers defence response in cotton.

Verticillium dahliae is a widespread and destructive soilborne vascular pathogenic fungus that causes serious diseases in dicot plants. Here, comparative transcriptome analysis showed that the number of genes upregulated in defoliating pathotype V991 was significantly higher than in the non-defoliating pathotype 1cd3-2 during the early response of cotton. Combined with analysis of the secretome during the V991-cotton interaction, an elicitor VP2 was identified, which was highly upregulated at the early stage of V991 invasion, but was barely expressed during the 1cd3-2-cotton interaction. Full-length VP2 could induce cell death in several plant species, and which was dependent on NbBAK1 but not on NbSOBIR1 in N. benthamiana. Knock-out of VP2 attenuated the pathogenicity of V991. Furthermore, overexpression of VP2 in cotton enhanced resistance to V. dahliae without causing abnormal plant growth and development. Several genes involved in JA, SA and lignin synthesis were significantly upregulated in VP2-overexpressing cotton. The contents of JA, SA, and lignin were also significantly higher than in the wild-type control. In summary, the identified elicitor VP2, recognized by the receptor in the plant membrane, triggers the cotton immune response and enhances disease resistance.

GhWRKY41 forms a positive feedback regulation loop and increases cotton defence response against Verticillium dahliae by regulating phenylpropanoid metabolism.

Despite the established significance of WRKY proteins and phenylpropanoid metabolism in plant immunity, how WRKY proteins modulate aspects of the phenylpropanoid pathway remains undetermined. To understand better the role of WRKY proteins in plant defence, we identified a cotton (Gossypium hirsutum) protein, GhWRKY41, that is, universally and rapidly induced in three disease-resistant cotton cultivars following inoculation with the plant pathogenic fungus, Verticillium dahliae. We show that overexpression of GhWRKY41 in transgenic cotton and Arabidopsis enhances resistance to V. dahliae, while knock-down increases cotton more susceptibility to the fungus. GhWRKY41 physically interacts with itself and directly activates its own transcription. A genome-wide chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), in combination with RNA sequencing (RNA-seq) analyses, revealed that 43.1% of GhWRKY41-binding genes were up-regulated in cotton upon inoculation with V. dahliae, including several phenylpropanoid metabolism master switches, receptor kinases, and disease resistance-related proteins. We also show that GhWRKY41 homodimer directly activates the expression of GhC4H and Gh4CL, thereby modulating the accumulation of lignin and flavonoids. This finding expands our understanding of WRKY-WRKY protein interactions and provides important insights into the regulation of the phenylpropanoid pathway in plant immune responses by a WRKY protein.

High temperature induces male sterility via MYB66-MYB4-Casein kinase I signaling in cotton.

High temperature (HT) causes male sterility and decreases crop yields. Our previous works have demonstrated that sugar and auxin signaling pathways, Gossypium hirsutum Casein kinase I (GhCKI), and DNA methylation are all involved in HT-induced male sterility in cotton. However, the signaling mechanisms leading to distinct GhCKI expression patterns induced by HT between HT-tolerant and HT-sensitive cotton anthers remain largely unknown. Here, we identified a GhCKI promoter (ProGhCKI) region that functions in response to HT in anthers and found the transcription factor GhMYB4 binds to this region to act as an upstream positive regulator of GhCKI. In the tapetum of early-stage cotton anthers, upregulated expression of GhMYB4 under HT and overexpressed GhMYB4 under normal temperature both led to severe male sterility phenotypes, coupled with enhanced expression of GhCKI. We also found that GhMYB4 interacts with GhMYB66 to form a heterodimer to enhance its binding to ProGhCKI. However, GhMYB66 showed an expression pattern similar to GhMYB4 under HT but did not directly bind to ProGhCKI. Furthermore, HT reduced siRNA-mediated CHH DNA methylations in the GhMYB4 promoter, which enhanced the expression of GhMYB4 in tetrad stage anthers and promoted the formation of the GhMYB4/GhMYB66 heterodimer, which in turn elevated the transcription of GhCKI in the tapetum, leading to male sterility. Overall, we shed light on the GhMYB66-GhMYB4-GhCKI regulatory pathway in response to HT in cotton anthers.

GhTULP34, a member of tubby-like proteins, interacts with GhSKP1A to negatively regulate plant osmotic stress.

Tubby-like protein genes (TULPs), present in the form of large multigene families, play important roles in environmental stress. However, little is known regarding the TULP family genes in cotton. In this study, we systematically identified and analyzed the membership, characterization, and evolutionary relationship of TULPs in four species of cotton. Transcriptome analysis indicated that GhTULPs participate in environmental stress and cotton tissue development. At the same time, we also predicted and analyzed the potential molecular regulatory mechanisms and functions of TULPs. GhTULP34, as a candidate gene, significantly reduced the germination rate of transgenic Arabidopsis plants under salt stress, and inhibited root development and stomatal closure under mannitol stress. The yeast two-hybrid and luciferase (LUC) systems showed that GhTULP34 can interact with GhSKP1A, a subunit of the SCF-type (Skp1-Cullin-1-F-box) complex. This study will provide a basis and reference for future research on their roles in stress tolerance.

Cytochrome P450 mono-oxygenase CYP703A2 plays a central role in sporopollenin formation and ms5ms6 fertility in cotton.

The double-recessive genic male-sterile (ms) line ms5 ms6 has been used to develop cotton (Gossypium hirsutum) hybrids for many years, but its molecular-genetic basis has remained unclear. Here, we identified the Ms5 and Ms6 loci through map-based cloning and confirmed their function in male sterility through CRISPR/Cas9 gene editing. Ms5 and Ms6 are highly expressed in stages 7-9 anthers and encode the cytochrome P450 mono-oxygenases CYP703A2-A and CYP703A2-D. The ms5 mutant carries a single-nucleotide C-to-T nonsense mutation leading to premature chain termination at amino acid 312 (GhCYP703A2-A312aa ), and ms6 carries three nonsynonymous substitutions (D98E, E168K, and G198R) and a synonymous mutation (L11L). Enzyme assays showed that GhCYP703A2 proteins hydroxylate fatty acids, and the ms5 (GhCYP703A2-A312aa ) and ms6 (GhCYP703A2-DD98E,E168K,G198R ) mutant proteins have decreased enzyme activities. Biochemical and lipidomic analyses showed that in ms5 ms6 plants, C12-C18 free fatty acid and phospholipid levels are significantly elevated in stages 7-9 anthers, while stages 8-10 anthers lack sporopollenin fluorescence around the pollen, causing microspore degradation and male sterility. Overall, our characterization uncovered functions of GhCYP703A2 in sporopollenin formation and fertility, providing guidance for creating male-sterile lines to facilitate hybrid cotton production and therefore exploit heterosis for improvement of cotton.

An enhanced photosynthesis and carbohydrate metabolic capability contributes to heterosis of the cotton (Gossypium hirsutum) hybrid 'Huaza Mian H318', as revealed by genome-wide gene expression analysis.

BACKGROUND: Heterosis has been exploited for decades in different crops due to resulting in dramatic increases in yield, but relatively little molecular evidence on this topic was reported in cotton. RESULTS: The elite cotton hybrid variety 'Huaza Mian H318' (H318) and its parental lines were used to explore the source of its yield heterosis. A four-year investigation of yield-related traits showed that the boll number of H318 showed higher stability than that of its two parents, both in suitable and unsuitable climate years. In addition, the hybrid H318 grew faster and showed higher fresh and dry weights than its parental lines at the seedling stage. Transcriptome analysis of seedlings identified 17,308 differentially expressed genes (DEGs) between H318 and its parental lines, and 3490 extremely changed DEGs were screened out for later analysis. Most DEGs (3472/3490) were gathered between H318 and its paternal line (4-5), and only 64 DEGs were found between H318 and its maternal line (B0011), which implied that H318 displays more similar transcriptional patterns to its maternal parent at the seedling stage. GO and KEGG analyses showed that these DEGs were highly enriched in photosynthesis, lipid metabolic, carbohydrate metabolic and oxidation-reduction processes, and the expression level of these DEGs was significantly higher in H318 relative to its parental lines, which implied that photosynthesis, metabolism and stress resistances were enhanced in H318. CONCLUSION: The enhanced photosynthesis, lipid and carbohydrate metabolic capabilities contribute to the heterosis of H318 at the seedling stage, and establishes a material foundation for subsequent higher boll-setting rates in complex field environments.

Genome-wide identification, evolutionary estimation and functional characterization of two cotton CKI gene types.

BACKGROUND: Casein kinase I (CKI) is a kind of serine/threonine protein kinase highly conserved in plants and animals. Although molecular function of individual member of CKI family has been investigated in Arabidopsis, little is known about their evolution and functions in Gossypium. RESULTS: In this study, five cotton species were applied to study CKI gene family in cotton, twenty-two species were applied to trace the origin and divergence of CKI genes. Four important insights were gained: (i) the cotton CKI genes were classified into two types based on their structural characteristics; (ii) two types of CKI genes expanded with tetraploid event in cotton; (iii) two types of CKI genes likely diverged about 1.5 billion years ago when red and green algae diverged; (iv) two types of cotton CKI genes which highly expressed in leaves showed stronger response to photoperiod (circadian clock) and light signal, and most two types of CKI genes highly expressed in anther showed identical heat inducible expression during anther development in tetraploid cotton (Gossypium hirsutum). CONCLUSION: This study provides genome-wide insights into the evolutionary history of cotton CKI genes and lays a foundation for further investigation of the functional differentiation of two types of CKI genes in specific developmental processes and environmental stress conditions.

A combination of genome-wide and transcriptome-wide association studies reveals genetic elements leading to male sterility during high temperature stress in cotton.

Global warming has reduced the productivity of many field-grown crops, as the effects of high temperatures can lead to male sterility in such plants. Genetic regulation of the high temperature (HT) response in the major crop cotton is poorly understood. We determined the functionality and transcriptomes of the anthers of 218 cotton accessions grown under HT stress. By analyzing transcriptome divergence and implementing a genome-wide association study (GWAS), we identified three thermal tolerance associated loci which contained 75 protein coding genes and 27 long noncoding RNAs, and provided expression quantitative trait loci (eQTLs) for 13 132 transcripts. A transcriptome-wide association study (TWAS) confirmed six causal elements for the HT response (three genes overlapped with the GWAS results) which are involved in protein kinase activity. The most susceptible gene, GhHRK1, was confirmed to be a previously uncharacterized negative regulator of the HT response in both cotton and Arabidopsis. These functional variants provide a new understanding of the genetic basis for HT tolerance in male reproductive organs.

Orchestration of plant development and defense by indirect crosstalk of salicylic acid and brassinosteorid signaling via transcription factor GhTINY2.

Salicylic acid (SA) and brassinosteroids (BRs) are well known to regulate diverse processes of plant development and stress responses, but the mechanisms by which these phytohormones mediate the growth and defense trade-off are largely unclear. In addition, little is known about the roles of DEHYDRATION RESPONSIVE ELEMENT BINDING transcription factors, especially in biotic stress and plant growth. Here, we identified a cotton (Gossypium hirsutum) APETALA2/ETHYLENE RESPONSIVE FACTOR gene GhTINY2 that is strongly induced by Verticillium dahliae. Overexpression of GhTINY2 in cotton and Arabidopsis enhanced tolerance to V. dahliae, while knockdown of expression increased the susceptibility of cotton to the pathogen. GhTINY2 was found to promote SA accumulation and SA signaling transduction by directly activating expression of WRKY51. Moreover, GhTINY2-overexpressing cotton and Arabidopsis showed retardation of growth, increased sensitivity to inhibitors of BR biosynthesis, down-regulation of several BR-induced genes, and up-regulation of BR-repressed genes, while GhTINY2-RNAi cotton showed the opposite effects. We further determined that GhTINY2 negatively regulates BR signaling by interacting with BRASSINAZOLE-RESISTANT 1 (BZR1) and restraining its transcriptional activation of the expression of INDOLE-3-ACETIC ACID INDUCIBLE 19 (IAA19). These findings indicate that GhTINY2 fine-tunes the trade-off between immunity and growth via indirect crosstalk between WRKY51-mediated SA biosynthesis and BZR1-IAA19-regulated BR signaling.

GhMYB4 downregulates lignin biosynthesis and enhances cotton resistance to Verticillium dahliae.

KEY MESSAGE: GhMYB4 acts as a negative regulator in lignin biosynthesis, which results in alteration of cell wall integrity and activation of cotton defense response. Verticillium wilt of cotton (Gossypium hirsutum) caused by the soil-borne fungus Verticillium dahliae (V. dahliae) represents one of the most important constraints of cotton production worldwide. Mining of the genes involved in disease resistance and illuminating the molecular mechanisms that underlie this resistance is of great importance in cotton breeding programs. Defense-induced lignification in plants is necessary for innate immunity, and there are reports of a correlation between increased lignification and disease resistance. In this study, we present an example in cotton whereby plants with reduced lignin content also exhibit enhanced disease resistance. We identified a negative regulator of lignin synthesis, in cotton encoded in GhMYB4. Overexpression of GhMYB4 in cotton and Arabidopsis enhanced resistance to V. dahliae  with reduced lignin deposition. Moreover, GhMYB4 could bind the promoters of several genes involved in lignin synthesis, such as GhC4H-1, GhC4H-2, Gh4CL-4, and GhCAD-3, and impair their expression. The reduction of lignin content in GhMYB4-overexpressing cotton led to alterations of cell wall integrity (CWI) and released more oligogalacturonides (OGs) which may act as damage-associated molecular patterns (DAMPs) to stimulate plant defense responses. In support of this hypothesis, exogenous application with polygalacturonic acid (PGA) in cotton activated biosynthesis of jasmonic acid (JA) and JA-mediated defense against V. dahliae, similar to that described for cotton plants overexpressing GhMYB4. This study provides a new candidate gene for cotton disease-resistant breeding and an increased understanding of the relationship between lignin synthesis, OG release, and plant immunity.

Suppression of tryptophan synthase activates cotton immunity by triggering cell death via promoting SA synthesis.

Primary metabolism plays an important role in plant growth and development, however the relationship between primary metabolism and the adaptive immune response is largely unknown. Here, we employed RNA interference (RNAi), virus-induced gene silencing (VIGS) technology, phytohormone profiling, genetic studies, and transcriptome and metabolome analysis to investigate the function of the tryptophan synthesis pathway in the resistance of cotton to V. dahliae. We found that knock-down of GbTSA1 (Tryptophan Synthase α) and GbTSB1 (tryptophan synthase ß) induced a spontaneous cell death phenotype in a salicylic acid (SA)-dependent manner and enhanced resistance to V. dahliae in cotton plants. Metabolome analysis showed that indole and indolic metabolites were highly accumulated in GbTSA1- or GbTSB1-silenced plants. Transcriptomic analysis showed that exogenous indole promotes the expression levels of genes involved in SA synthesis and the defense response. Similarly, indole application strongly enhanced cotton resistance to V. dahliae. These results suggested that metabolic intermediates in the Trp synthesis pathway may be a signal to activate SA synthesis. These results also provided a strategy to elicit plant defense responses by the application of indole.

Deficiencies in the formation and regulation of anther cuticle and tryphine contribute to male sterility in cotton PGMS line.

BACKGROUND: Male sterility is a simple and efficient pollination control system that is widely exploited in hybrid breeding. In upland cotton, CCRI9106, a photosensitive genetic male sterile (PGMS) mutant isolated from CCRI040029, was reported of great advantages to cotton heterosis. However, little information concerning the male sterility of CCRI9106 is known. Here, comparative transcriptome analysis of CCRI9106 (the mutant, MT) and CCRI040029 (the wild type, WT) anthers in Anyang (long-day, male sterile condition to CCRI9106) was performed to reveal the potential male sterile mechanism of CCRI9106. RESULTS: Light and electron microscopy revealed that the male sterility phenotype of MT was mainly attributed to irregularly exine, lacking tryphine and immature anther cuticle. Based on the cytological characteristics of MT anthers, anther RNA libraries (18 in total) of tetrad (TTP), late uninucleate (lUNP) and binucleate (BNP) stages in MT and WT were constructed for transcriptomic analysis, therefore revealing a total of 870,4 differentially expressed genes (DEGs). By performing gene expression pattern analysis and protein-protein interaction (PPI) networks construction, we found down-regulation of DEGs, which enriched by the lipid biosynthetic process and the synthesis pathways of several types of secondary metabolites such as terpenoids, flavonoids and steroids, may crucial to the male sterility phenotype of MT, and resulting in the defects of anther cuticle and tryphine, even the irregularly exine. Furthermore, several lipid-related genes together with ABA-related genes and MYB transcription factors were identified as hub genes via weighted gene co-expression network analysis (WGCNA). Additionally, the ABA content of MT anthers was reduced across all stages when compared with WT anthers. At last, genes related to the formation of anther cuticle and tryphine could activated in MT under short-day condition. CONCLUSIONS: We propose that the down-regulation of genes related to the assembly of anther cuticle and tryphine may lead to the male sterile phenotype of MT, and MYB transcription factors together with ABA played key regulatory roles in these processes. The conversion of fertility in different photoperiods may closely relate to the functional expression of these genes. These findings contribute to elucidate the mechanism of male sterility in upland cotton.

ABA signaling is negatively regulated by GbWRKY1 through JAZ1 and ABI1 to affect salt and drought tolerance.

KEY MESSAGE: GbWRKY1 can function as a negative regulator of ABA signaling via JAZ1 and ABI1, with effects on salt and drought tolerance. WRKY transcription factors play important roles in plant development and stress responses. GbWRKY1 was initially identified as a defense-related gene in cotton and negatively regulates the response to fungal pathogens by activating the expression of JAZ1. Here, we characterized the role of GbWRKY1, an orthologue of the Arabidopsis gene AtWRKY75, in abiotic stress (salt and drought) and established novel connection between JAZ1 and ABA signaling in Arabidopsis. GbWRKY1 is nucleus localized and its expression is significantly induced by treatment with ABA and osmotic stresses NaCl and PEG. Increased levels of expression of GbWRKY1 in transgenic Arabidopsis enhance sensitivity to salt and drought as revealed by seed germination tests and soil stress experiments. Similarly, GbWRKY1 overexpression cotton plants also display increased sensitivity to PEG treatment and drought. Expression analysis shows that the induction of two ABA responsive genes, RAB18 and RD29A by NaCl, mannitol, and ABA treatment is significantly impaired in GbWRKY1 overexpression Arabidopsis lines. GbWRKY1 overexpression Arabidopsis displays a strong ABA-insensitive phenotype at both germination and early stages of seedling development. Further genetic evidence suggested that the ABA-insensitive phenotype of GbWRKY1 overexpression Arabidopsis was dependent on JAZ1, and overexpression of JAZ1 also displayed an ABA-insensitive phenotype. In addition, yeast two hybrid and bimolecular fluorescence complementation assays showed that JAZ1 directly interacts with ABI1, a key negative regulator of ABA signaling. We, therefore, demonstrate that GbWRKY1 acts as a negative regulator of ABA signaling, through an interaction network involving JAZ1 and ABI1, to regulate salt and drought tolerance.

GhCyP3 improves the resistance of cotton to Verticillium dahliae by inhibiting the E3 ubiquitin ligase activity of GhPUB17.

KEY MESSAGE: A U-box E3 ubiquitin ligase GhPUB17 is inhibited by GhCyP3 with antifungal activity and acts as a negative regulator involved in cotton resistance to Verticillium dahliae. E3 ubiquitin ligases, the key component enzymes of the ubiquitin-proteasome system, which contains the most diverse structural and functional members involved in the determination of target specificity and the regulation of metabolism, have been well documented in previous studies. Here, we identify GhPUB17, a U-box E3 ligase in cotton that has ubiquitination activity and is involved in the cotton immune response to Verticillium dahliae. The expression level of GhPUB17 is downregulated in the ssn mutant with a constitutively activated immune response (Sun et al., Nat Commun 5:5372, 2014). Infection with V. dahliae or exogenous hormone treatment, including jasmonic acid and salicylic acid, significantly upregulated GhPUB17 in cotton roots, which suggested a possible role for this E3 ligase in the plant immune response to pathogens. Moreover, GhPUB17-knockdown cotton plants are more resistant to V. dahliae, whereas GhPUB17-overexpressing plants are more susceptible to the pathogen, which indicated that GhPUB17 is a negative regulator of cotton resistance to V. dahliae. A yeast two-hybrid (Y2H) assay identified GhCyP3 as a protein that interacts with GhPUB17, and this finding was confirmed by further protein interaction assays. The downregulation of GhCyP3 in cotton seedlings attenuated the plants' resistance to V. dahliae. In addition, GhCyP3 showed antifungal activity against V. dahliae, and the E3 ligase activity of GhPUB17 was repressed by GhCyP3 in vitro. These results suggest that GhPUB17 negatively regulates cotton immunity to V. dahliae and that the antifungal protein GhCyP3 likely interacts with and inhibits the ligase activity of GhPUB17 and plays an important role in the cotton-Verticillium interaction.

GbSOBIR1 confers Verticillium wilt resistance by phosphorylating the transcriptional factor GbbHLH171 in Gossypium barbadense.

Receptor-like kinases (RLKs) are important components of plant innate immunity. Although recent studies have revealed that the RLK suppressor of BIR1-1 (SOBIR1) can interact with multiple receptor-like proteins and is required for resistance against fungal pathogens, how the signal is transduced and triggers immune responses remains enigmatic. In this study, we identified a defence-related RLK from Gossypium barbadense (designated GbSOBIR1) and investigated its functional mechanism. Expression of the GbSOBIR1 gene is ubiquitous in cotton plants and is induced by Verticillium dahliae inoculation. Knock-down of GbSOBIR1 by virus-induced gene silencing resulted in attenuated resistance of cotton plants to V. dahliae, while heterologous overexpression of GbSOBIR1 in Arabidopsis improves resistance. We also found that the kinase region of GbSOBIR1 interacts with a basic helix-loop-helix (bHLH) transcription factor identified as GbbHLH171 in a yeast-two-hybrid screen. GbbHLH171 could interact with and be phosphorylated by GbSOBIR1 in vitro and in vivo and contributes positively to the resistance of cotton against V. dahliae. Furthermore, we found that this phosphorylation is essential to the transcriptional activity and functional role of GbbHLH171. We also show by spectrometric analysis and site-directed mutagenesis that Ser413 is the GbSOBIR1-mediated phosphorylation site of GbbHLH171. These results demonstrate that GbSOBIR1 interacts with GbbHLH171 and plays a critical role in cotton resistance to V. dahliae.

GhCPK33 Negatively Regulates Defense against Verticillium dahliae by Phosphorylating GhOPR3.

Verticillium wilt, caused by the soil-borne fungus Verticillium dahliae, is a destructive vascular disease in plants. Approximately 200 dicotyledonous plant species in temperate and subtropical regions are susceptible to this notorious pathogen. Previous studies showed that jasmonic acid (JA) plays a crucial role in plant-V. dahliae interactions. V. dahliae infection generally induces significant JA accumulation in local and distal tissues of the plant. Although JA biosynthesis and the associated enzymes have been studied intensively, the precise mechanism regulating JA biosynthesis upon V. dahliae infection remains unknown. Here, we identified the calcium-dependent protein kinase GhCPK33 from upland cotton (Gossypium hirsutum) as a negative regulator of resistance to V. dahliae that directly manipulates JA biosynthesis. Knockdown of GhCPK33 by Agrobacterium tumefaciens-mediated virus-induced gene silencing constitutively activated JA biosynthesis and JA-mediated defense responses and enhanced resistance to V dahliae Further analysis revealed that GhCPK33 interacts with 12-oxophytodienoate reductase3 (GhOPR3) in peroxisomes. GhCPK33 phosphorylates GhOPR3 at threonine-246, leading to decreased stability of GhOPR3, which consequently limits JA biosynthesis. We propose that GhCPK33 is a potential molecular target for improving resistance to Verticillium wilt disease in cotton.

Laccase GhLac1 Modulates Broad-Spectrum Biotic Stress Tolerance via Manipulating Phenylpropanoid Pathway and Jasmonic Acid Synthesis.

Plants are constantly challenged by a multitude of pathogens and pests, which causes massive yield and quality losses annually. A promising approach to reduce such losses is to enhance the immune system of plants through genetic engineering. Previous work has shown that laccases (p-diphenol:dioxygen oxidoreductase, EC 1.10.3.2) function as lignin polymerization enzymes. Here we demonstrate that transgenic manipulation of the expression of the laccase gene GhLac1 in cotton (Gossypium hirsutum) can confer an enhanced defense response to both pathogens and pests. Overexpression of GhLac1 leads to increased lignification, associated with increased tolerance to the fungal pathogen Verticillium dahliae and to the insect pests cotton bollworm (Helicoverpa armigera) and cotton aphid (Aphis gosypii). Suppression of GhLac1 expression leads to a redirection of metabolic flux in the phenylpropanoid pathway, causing the accumulation of JA and secondary metabolites that confer resistance to V. dahliae and cotton bollworm; it also leads to increased susceptibility to cotton aphid. Plant laccases therefore provide a new molecular tool to engineer pest and pathogen resistance in crops.

microRNAs involved in auxin signalling modulate male sterility under high-temperature stress in cotton (Gossypium hirsutum).

Male sterility caused by long-term high-temperature (HT) stress occurs widely in crops. MicroRNAs (miRNAs), a class of endogenous non-coding small RNAs, play an important role in the plant response to various abiotic stresses. To dissect the working principle of miRNAs in male sterility under HT stress in cotton, a total of 112 known miRNAs, 270 novel miRNAs and 347 target genes were identified from anthers of HT-insensitive (84021) and HT-sensitive (H05) cotton cultivars under normal-temperature and HT conditions through small RNA and degradome sequencing. Quantitative reverse transcriptase-polymerase chain reaction and 5'-RNA ligase-mediated rapid amplification of cDNA ends experiments were used to validate the sequencing data. The results show that miR156 was suppressed by HT stress in both 84021 and H05; miR160 was suppressed in 84021 but induced in H05. Correspondingly, SPLs (target genes of miR156) were induced both in 84021 and H05; ARF10 and ARF17 (target genes of miR160) were induced in 84021 but suppressed in H05. Overexpressing miR160 increased cotton sensitivity to HT stress seen as anther indehiscence, associated with the suppression of ARF10 and ARF17 expression, thereby activating the auxin response that leads to anther indehiscence. Supporting this role for auxin, exogenous Indole-3-acetic acid (IAA) leads to a stronger male sterility phenotype both in 84021 and H05 under HT stress. Cotton plants overexpressing miR157 suppressed the auxin signal, and also showed enhanced sensitivity to HT stress, with microspore abortion and anther indehiscence. Thus, we propose that the auxin signal, mediated by miRNAs, is essential for cotton anther fertility under HT stress.

Genome-wide identification of lipoxygenase gene family in cotton and functional characterization in response to abiotic stresses.

BACKGROUND: Plant lipoxygenase (LOX) genes are members of the non-haeme iron-containing dioxygenase family that catalyze the oxidation of polyunsaturated fatty acids into functionally diverse oxylipins. The LOX family genes have been extensively studied under biotic and abiotic stresses, both in model and non-model plant species; however, information on their roles in cotton is still limited. RESULTS: A total of 64 putative LOX genes were identified in four cotton species (Gossypium (G. hirsutum, G. barbadense, G. arboreum, and G. raimondii)). In the phylogenetic tree, these genes were clustered into three categories (9-LOX, 13-LOX type I, and 13-LOX type II). Segmental duplication of putative LOX genes was observed between homologues from A2 to At and D5 to Dt hinting at allopolyploidy in cultivated tetraploid species (G. hirsutum and G. barbadense). The structure and motif composition of GhLOX genes appears to be relatively conserved among the subfamilies. Moreover, many cis-acting elements related to growth, stresses, and phytohormone signaling were found in the promoter regions of GhLOX genes. Gene expression analysis revealed that all GhLOX genes were induced in at least two tissues and the majority of GhLOX genes were up-regulated in response to heat and salinity stress. Specific expressions of some genes in response to exogenous phytohormones suggest their potential roles in regulating growth and stress responses. In addition, functional characterization of two candidate genes (GhLOX12 and GhLOX13) using virus induced gene silencing (VIGS) approach revealed their increased sensitivity to salinity stress in target gene-silenced cotton. Compared with controls, target gene-silenced plants showed significantly higher chlorophyll degradation, higher H2O2, malondialdehyde (MDA) and proline accumulation but significantly reduced superoxide dismutase (SOD) activity, suggesting their reduced ability to effectively degrade accumulated reactive oxygen species (ROS). CONCLUSION: This genome-wide study provides a systematic analysis of the cotton LOX gene family using bioinformatics tools. Differential expression patterns of cotton LOX genes in different tissues and under various abiotic stress conditions provide insights towards understanding the potential functions of candidate genes. Beyond the findings reported here, our study provides a basis for further uncovering the biological roles of LOX genes in cotton development and adaptation to stress conditions.