Cotton ethylene response factor Gh ERF91 is involved in the defense against Verticillium dahliae

Verticillium dahliae causes significant losses in cotton production.To reveal the mechanism of the defense response to V.dahliae in cotton,transcriptomic analyses were performed using cotton cultivars M138(V.dahliae-resistant)and P2(V.dahliae-susceptible).The results revealed 11,076 and 6,640 differentially expressed genes(DEGs)in response to V.dahliae,respectively.The weighted gene co-expression network analysis of 4,633 transcription factors(TFs)indicated a“MEblue”module containing 654 TFs that strongly correlate with resistance to V.dahliae.Among these TFs,the ethylene response factor Ghi_A05G10166(GhERF91)was identified as a putative hub gene with a defense response against V.dahliae.A virus-induced gene silencing assay and exogenous application of ethephon showed that GhERF91 is activated by ethylene and positively regulates the response to V.dahliae exposure in cotton.This study provides fundamental transcriptome data and a putative causal gene(GhERF91)associated with resistance to V.dahliae,as well as genetic resources for breeding V.dahliae-resistant cotton.

Apical meristem transcriptome analysis identifies a role for the blue light receptor gene GhFKF1 in cotton architecture development

Cotton architecture is determined by the differentiation fate transition of axillary meristem(AM),and influences cotton yield and the efficiency of mechanized harvesting.We observed that the initiation of flowering primordium was earlier in early-maturing than that in late-maturing cultivars during the differentiation and development of AM.The RNA-Seq and expression level analyses showed that genes FLAVIN BINDING,KELCH REPEAT,F-BOX1(GhFKF1),and GIGANTEA(GhGI)were in response to circadian rhythms,and involved in the regulation of cotton flowering.The gene structure,predicted protein structure,and motif content analyses showed that in Arabidopsis,cotton,rapseed,and soybean,proteins GhFKF1 and GhGI were functionally conserved and share evolutionary origins.Compared to the wild type,in GhFKF1 mutants that were created by the CRISPR/Cas9 system,the initiation of branch primordium was inhibited.Conversely,the knocking out of GhGI increased the number of AM differentiating into flower primordium,and there were much more lateral branch differentiation and development.Besides,we investigated that proteins GhFKF1 and GhGI can interact with each other.These results suggest that GhFKF1 and GhGI are key regulators of cotton architecture development,and may collaborate to regulate the differentiation fate transition of AM,ultimately influencing plant architecture.We describe a strategy for using the CRISPR/Cas9 system to increase cotton adaptation and productivity by optimizing plant architecture.

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.

Identification of candidate genes for aphid resistance in upland cotton by QTL mapping and expression analysis

Lignin is one of the main components of cell walls and is essential for resistance to insect pests in plants.Cotton plants are damaged by aphid(Aphis gossypii) worldwide but resistant breeding is undeveloped due to scarce knowledge on resistance genes and the mechanism. This study reported a lignin biosynthesisrelated gene identified in the F_(2) population derived from the cross between cotton cultivars Xinluzao 61(resistant to aphid) and Xinluzao 50(susceptible to aphid). A quantitative trait locus was mapped on chromosome D04 with a logarithm of odds(LOD) score of 5.99 and phenotypic effect of 27%. RNA-seq analysis of candidate intervals showed that the expression level of GH_D04G1418 was higher in the resistant cultivar than in the susceptible cultivar. This locus is close to AtLAC4 in the phylogenetic tree and contains a conserved laccase domain. Hence, it was designated GhLAC4-3. Silencing of GhLAC4-3 in Xinluzao 61 via virus-induced gene silencing(VIGS) resulted in decreased lignin content and increased susceptibility to aphids. These results suggest that GhLAC4-3 might enhance aphid resistance by regulating lignin biosynthesis in cotton.

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.