High-temperature stress suppresses allene oxide cyclase 2 and causes male sterility in cotton by disrupting jasmonic acid signaling

Cotton(Gossypium spp.) yield is reduced by stress. In this study, high temperature(HT) suppressed the expression of the jasmonic acid(JA) biosynthesis gene allene oxide cyclase 2(GhAOC2), reducing JA content and causing male sterility in the cotton HT-sensitive line H05. Anther sterility was reversed by exogenous application of methyl jasmonate(MeJA) to early buds. To elucidate the role of GhAOC2 in JA biosynthesis and identify its putative contribution to the anther response to HT, we created gene knockout cotton plants using the CRISPR/Cas9 system. Ghaoc2 mutant lines showed male-sterile flowers with reduced JA content in the anthers at the tetrad stage(TS), tapetum degradation stage(TDS), and anther dehiscence stage(ADS). Exogenous application of MeJA to early mutant buds(containing TS or TDS anthers) rescued the sterile pollen and indehiscent anther phenotypes, while ROS signals were reduced in ADS anthers. We propose that HT downregulates the expression of GhAOC2 in anthers, reducing JA biosynthesis and causing excessive ROS accumulation in anthers, leading to male sterility. These findings suggest exogenous JA application as a strategy for increasing male fertility in cotton under HT.

Attenuation of ethylene signaling increases cotton resistance to a defoliating strain of Verticillium dahliae

The severity of Verticillium wilt on cotton caused by defoliating strains of Verticillium dahliae has gradually increased and threatens production worldwide. Identification of the molecular components of leaf defoliation may increase cotton tolerance to V. dahliae. Ethylene, a major player in plant physiological processes, is often associated with senescence and defoliation of plants. We investigated the cotton–V.dahliae interaction with a focus on the role of ethylene in defoliation and defense against V. dahliae.Cotton plants inoculated with V. dahliae isolate V991, a defoliating strain, accumulated more ethylene and showed increased disease symptoms than those inoculated with a non-defoliating strain. In cotton with a transiently silenced ethylene synthesis gene(GhACOs) and signaling gene(GhEINs) during cotton–V. dahliae interaction, ethylene produced was derived from cotton and more ethylene increased cotton susceptibility and defoliation rate. Overexpression of AtCTR1, a negative regulator in ethylene signaling, in cotton reduced sensitivity to ethylene and increased plant resistance to V. dahliae.Collectively, the results indicated precise regulation of ethylene synthesis or signaling pathways improve cotton resistant to Verticillium wilt.

miR398b negatively regulates cotton immune responses to Verticillium dahliae via multiple targets

MicroRNAs(miRNAs)play essential roles in plant defense responses,although such roles have not been identified in cotton in response to the plant pathogenic fungus Verticillium dahliae.In this study,the functions of miR398b and its target genes in cotton-V.dahliae interaction were investigated.The transcript levels of miR398b were down-regulated by V.dahliae infection and miR398b overexpression in cotton made the plants more susceptible to V.dahliae.The results suggest that miR398b negatively regulates cotton resistance to V.dahliae.This may occur by miR398b repression of some CC-NBS-LRR genes via translational inhibition,interfering with defense responses and leading to cotton susceptibility to V.dahliae.Alternatively,miR398b may guide the cleavage of the mRNAs of GhCSD1,GhCSD2 and GhCCS,each of which functions in reactive oxygen species(ROS)regulation and homeostasis,thereby causing excessive ROS accumulation in miR398b-overexpressing plants in response to V.dahliae infection.This study suggests conserved and novel roles of miR398b in the cotton–V.dahliae interaction.These discoveries may be coupled with new strategies in cotton breeding programs to improve resistance to V.dahliae.

Histone H3 lysine 27 trimethylation suppresses jasmonate biosynthesis and signaling to affect male fertility under high temperature in cotton

High-temperature(HT)stress causes male sterility in crops,thus decreasing yields.To explore the possible contribution of histone modifications to male fertility under HT conditions,we defined the histone methylation landscape for the marks histone H3 lysine 27 trimethylation(H3K27me3)and histone H3 lysine 4 trimethylation(H3K4me3)by chromatin immunoprecipitation sequencing(ChIP-seq)in two differing upland cotton(Gossypium hirsutum)varieties.We observed a global disruption in H3K4me3 and H3K27me3 modifications,especially H3K27me3,in cotton anthers subjected to HT.HT affected the bivalent H3K4me3–H3K27me3 modification more than either monovalent modification.We determined that removal of H3K27me3 at the promoters of jasmonate-related genes increased their expression,maintaining male fertility under HT in the HT-tolerant variety at the anther dehiscence stage.Modulating jasmonate homeostasis or signaling resulted in an anther indehiscence phenotype under HT.Chemical suppression of H3K27me3 deposition increased jasmonic acid contents and maintained male fertility under HT.In summary,our study provides new insights into the regulation of male fertility by histone modifications under HT and suggests a potential strategy for improving cotton HT tolerance.

A Novel Intelligent System for Dynamic Observation of Cotton Verticillium Wilt

Verticillium wilt is one of the most critical cotton diseases,which is widely distributed in cotton-producing countries.However,the conventional method of verticillium wilt investigation is still manual,which has the disadvantages of subjectivity and low efficiency.In this research,an intelligent vision-based system was proposed to dynamically observe cotton verticillium wilt with high accuracy and high throughput.Firstly,a 3-coordinate motion platform was designed with the movement range 6,100 mm×950 mm×500 mm,and a specific control unit was adopted to achieve accurate movement and automatic imaging.Secondly,the verticillium wilt recognition was established based on 6 deep learning models,in which the VarifocalNet(VFNet)model had the best performance with a mean average precision(mAP)of 0.932.Meanwhile,deformable convolution,deformable region of interest pooling,and soft non-maximum suppression optimization methods were adopted to improve VFNet,and the mAP of the VFNet-Improved model improved by 1.8%.The precision–recall curves showed that VFNet-Improved was superior to VFNet for each category and had a better improvement effect on the ill leaf category than fine leaf.The regression results showed that the system measurement based on VFNet-Improved achieved high consistency with manual measurements.Finally,the user software was designed based on VFNet-Improved,and the dynamic observation results proved that this system was able to accurately investigate cotton verticillium wilt and quantify the prevalence rate of different resistant varieties.In conclusion,this study has demonstrated a novel intelligent system for the dynamic observation of cotton verticillium wilt on the seedbed,which provides a feasible and effective tool for cotton breeding and disease resistance research.

Developing an efficient CRISPR-dCas9-TVderived transcriptional activation system to create three novel cotton germplasm materials

Dear Editor,With the development of CRISPR-Cas technology,an RNAguided CRISPR activation system has been developed in plants,in which a dead Cas9(dCas9)nuclease is fused to transcriptional activators to regulate the transcription of endogenous genes(Li et al.,2017;Liu et al.,2017;Manghwar et al.,2020;Ming et al.,2020;Pan et al.,2021).Precise upregulation of gene transcription is emerging as a promising approach for functional genomics research,molecular breeding,and germplasm innovation.However,there have been no reports on the use of the CRISPR-dCas9 transcriptional activation system in cotton.

Suppression of the homeobox gene HDTF_1 enhances resistance to Verticillium dahliae and Botrytis cinerea in cotton

Development of pathogen-resistant crops, such as fungus-resistant cotton, has significantly reduced chemical application and improved crop yield and quality. However, the mechanism of resistance to cotton pathogens such as Verticillium dahliae is still poorly understood. In this study, we characterized a cotton gene （HDTF1） that was isolated following transcriptome profiling during the resistance response of cotton to V. dahliae. HDTFI putatively encodes a homeodomain transcription factor, and its expression was found to be down-regulated in cotton upon inoculation with V. dahliae and Botrytis cinerea. To characterise the involvement of HDTF1 in the response to these pathogens, we used virusinduced gene silencing （VlGS） to generate HDTFl-silenced cotton. VIGS reduction in HDTF1 expression significantly enhanced cotton plant resistance to both pathogens. HDTF1 silencing resulted in activation of jasmonic acid （JA）-mediated signaling and JA accumulation. However, the silenced plants were not altered in the accumulation of salicylic acid （SA） or the expression of marker genes associated with SA signaling. These results suggest that HDTF1 is a negative regulator of the JA pathway, and resistance to V. dahliae and B. cinerea can be engineered by activation of JA signaling.

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-A^(312aa)),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-A^(312aa))and ms6(GhCYP703A2-D^(D98E,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.