Hongmei Wang

Background: Verticillium wilt (VW), caused by the fungal pathogen Verticillium dahliae, is a destructive disease that severely compromises cotton yield and fiber quality. Pyrimidine nucleotides, as essential metabolites and nucleic acid components, play critical roles in plant development and stress responses. However, genes involved in pyrimidine metabolism, especially their roles in disease resistance, remain largely uncharacterized in plants. Methods: Ghir_D05G039120, a gene encoding uridine kinase, shown to be associated with VW resistance in our previous study, was cloned and named as GhUKL4. The differential expression of GhUKL4 between the resistant and susceptible cultivars at multiple time points post-inoculation with V. dahliae was analyzed by quantitative real-time PCR (qRT-PCR), and the uracil phosphoribosyl transferase (UPRT) and uridine 5′-monophosphate kinase (UMPK) domains were verified by analyzing the amino acid sequences of GhUKL4. The role of GhUKL4 in the defense against VW infection was estimated by silencing GhUKL4 in the resistant and susceptible cultivars using virus-induced gene silencing (VIGS) analysis. Results: There were significant differences in the expression level of Ghir_D05G039120/ GhUKL4 among resistant and susceptible cotton lines. GhUKL4 contains UPRTase and UMPK domains, and there was one SNP between the resistant and susceptible cultivars in its 3′-UTR region. The silencing of GhUKL4 reduced cotton’s resistance to VW through mediating hormone signaling (JA) and oxidative stress (ROS) pathways. Conclusions: GhUKL4, encoding UMPK and UPRTase domain proteins, is a new regulatory factor associated with VW resistance in Gossypium hirsutum through fine-tuning JA-signalling and ROS bursting.

Climate deterioration, water shortages, and abiotic stress are the main threats worldwide that seriously affect cotton growth, yield, and fiber quality. Therefore, research on improving cotton yield and tolerance to biotic and abiotic stresses is of great importance. The NAC proteins are crucial and plant-specific transcription factors (TFs) that are involved in cotton growth, development, and stress responses. The comprehensive utilization of cotton NAC TFs in the improvement of cotton varieties through novel biotechnological methods is feasible. Based on cotton genomic data, genome-wide identification and analyses have revealed potential functions of cotton NAC genes. Here, we comprehensively summarize the recent progress in understanding cotton NAC TFs roles in regulating responses to drought, salt, and Verticillium wilt-related stresses, as well as leaf senescence and the development of fibers, xylem, and glands. The detailed regulatory network of NAC proteins in cotton is also elucidated. Cotton NAC TFs directly bind to the promoters of genes associated with ABA biosynthesis and secondary cell-wall formation, participate in several biological processes by interacting with related proteins, and regulate the expression of downstream genes. Studies have shown that the overexpression of NAC TF genes in cotton and other model plants improve their drought or salt tolerance. This review elucidates the latest findings on the functions and regulation of cotton NAC proteins, broadens our understanding of cotton NAC TFs, and lays a fundamental foundation for further molecular breeding research in cotton.

Background: Cotton is extremely affected by severe natural stresses. Drought is one of the most serious abiotic stress that adversely influences cotton growth, productivity, and fiber quality. Previous studies indicate that basic leucine-zipper (bZIP) transcription factors are involved in the response of plants to various stresses. However, the molecular function and regulatory mechanism of GhVIP1 in response to drought stress are still unknown. Results: In this research, GhVIP1 was cloned from a drought-tolerant variety. Expression of GhVIP1 was up-regulated in response to multiple abiotic stresses, especially under drought stress. And GhVIP1 was highly expressed in the root, stem, and 10 days post-anthesis ovule. Inhibiting the expression of GhVIP1 in cotton using the virus-induced gene silencing method resulted in higher electrical conductivity in leaves, but lower water content under drought stress compared with the WT plant. Overexpression of GhVIP1 in Arabidopsis enhanced plant drought tolerance through increasing the seed germination rate and improving the development of root. The exogenous expression of GhVIP1 up-regulated the transcription of genes associated with drought response and proline biosynthesis during drought stress in Arabidopsis. Conclusion: In summary, these results indicated that GhVIP1 played a positive role in plants' response to drought stress. The use of GhVIP1 via modern biotechnology might facilitate the improvement of drought tolerance in cotton cultivars.

Long intergenic non-coding RNAs (lincRNAs) have been demonstrated to be vital regulators of diverse biological processes in both animals and plants. While many lincRNAs have been identified in cotton, we still know little about the repositories and conservativeness of lincRNAs in different cotton species or about their role in responding to biotic stresses. Here, by using publicly available RNA-seq datasets from diverse sources, including experiments of Verticillium dahliae (Vd) infection, we identified 24,425 and 17,713 lincRNAs, respectively, in Gossypium hirsutum (Ghr) and G. barbadense (Gba), the two cultivated allotetraploid cotton species, and 6933 and 5911 lincRNAs, respectively, in G. arboreum (Gar) and G. raimondii (Gra), the two extant diploid progenitors of the allotetraploid cotton. While closely related subgenomes, such as Ghr_At and Gba_At, tend to have more conserved lincRNAs, most lincRNAs are species-specific. The majority of the synthetic and transcribed lincRNAs (78.2%) have a one-to-one orthologous relationship between different (sub)genomes, although a few of them (0.7%) are retained in all (sub)genomes of the four species. The Vd responsiveness of lincRNAs seems to be positively associated with their conservation level. The major functionalities of the Vd-responsive lincRNAs seem to be largely conserved amongst Gra, Ghr, and Gba. Many Vd-responsive Ghr-lincRNAs overlap with Vd-responsive QTL, and several lincRNAs were predicted to be endogenous target mimicries of miR482/2118, with a pair being highly conserved between Ghr and Gba. On top of the confirmation of the feature characteristics of the lincRNAs previously reported in cotton and other species, our study provided new insights into the conservativeness and divergence of lincRNAs during cotton evolution and into the relationship between the conservativeness and Vd responsiveness of lincRNAs. The study also identified candidate lincRNAs with a potential role in disease response for functional characterization.

Cotton is a globally important cash crop. Verticillium wilt (VW) is commonly known as “cancer” of cotton and causes serious loss of yield and fiber quality in cotton production around the world. Here, we performed a BSA-seq analysis using an F_2:3 segregation population to identify the candidate loci involved in VW resistance. Two QTLs (qvw-D05-1 and qvw-D05-2) related to VW resistance in cotton were identified using two resistant/susceptible bulks from the F₂ segregation population constructed by crossing the resistant cultivar ZZM2 with the susceptible cultivar J11. A total of 30stop-lost SNPs and 42 stop-gained SNPs, which included 17 genes, were screened in the qvw-D05-2 region by SnpEff analysis. Further analysis of the transcriptome data and qRT-PCR revealed that the expression level of Ghir_D05G037630 (designated as GhDRP) varied significantly at certain time points after infection with V. dahliae. The virus-induced gene silencing of GhDRP resulted in higher susceptibility of the plants to V. dahliae than the control, suggesting that GhDRP is involved in the resistance to V. dahlia infection. This study provides a method for rapid mining of quantitative trait loci and screening of candidate genes, as well as enriches the genomic information and gene resources for the molecular breeding of disease resistance in cotton.

The Negative on TATA-less (NOT) 2_3_5 domain proteins play key roles in mRNA metabolism and transcription regulation, but few comprehensive studies have focused on this protein family in plants. In our study, a total of 30 NOT2_3_5 genes were identified in four cotton genomes: Gossypium. arboretum, G. raimondii, G. hirsutum and G. barbadense. Phylogenetic analysis showed that all the NOT2_3_5 domain proteins were divided into two classes. The NOT2_3_5 genes were expanded frequently, and segmental duplication had significant effects in their expansion process. The cis-regulatory elements analysis of NOT2_3_5 promoter regions indicated that NOT2_3_5 domain proteins might participate in plant growth and development processes and responds to exogenous stimuli. Expression patterns demonstrated that all of the GhNOT2_3_5 genes were expressed in the majority of tissues and fiber development stages, and that these genes were induced by multiple stresses. Quantitative real-time PCR showed that GbNOT2_3_5 genes were up-regulated in response to verticillium wilt and the silencing of GbNOT2_3_5-3/8 and GbNOT2_3_5-4/9 led to more susceptibility to verticillium wilt than controls. Identification and analysis of the NOT2_3_5 protein family will be beneficial for further research on their biological functions.

Key message Combining GWAS, QTL-seq and transcriptome sequencing detected basal defense-related genes showing gDNA sequence variation and expression difference in diverse cotton lines, which might be the molecular mechanisms of VW resistance in G. hirsutum. Abstract Verticillium wilt (VW), which is caused by the soil-borne fungus Verticillium dahliae, is a major disease in cotton (Gossypim hirsutum) worldwide. To facilitate the understanding of the genetic basis for VW resistance in cotton, a genome-wide association study (GWAS), QTL-seq and transcriptome sequencing were performed. The GWAS of VW resistance in a panel of 120 core elite cotton accessions using the Cotton 63K Illumina Infinium SNP array identified 5 QTL from 18 significant SNPs meeting the 5% false discovery rate threshold on 5 chromosomes. All QTL identified through GWAS were found to be overlapped with previously reported QTL. By combining GWAS, QTL-seq and transcriptome sequencing, we identified eight candidate genes showing both gDNA sequence variation and expression difference between resistant and susceptible lines, most related to transcription factors (TFs), flavonoid biosynthesis and those involving in the plant basal defense and broad-spectrum disease resistance. Ten KASP markers were successfully validated in diverse cotton lines and could be deployed in marker-assisted breeding to enhance VW resistance. These results supported our inference that the gDNA sequence variation or expression difference of those genes involving in the basal defense in diverse cotton lines might be the molecular mechanisms of VW resistance in G. hirsutum.

The homologous leucine zipper/EF-hand-containing transmembranes (LETMs) are highly conserved across a broad range of eukaryotic organisms. The LETM functional characteristics involved in biological process have been identified primarily in animals, but little is known about the LETM biological function mode in plants. Based on the results of the current investigation, the GhLETM1 gene crucially affects filament elongation and anther dehiscence of the stamen in cotton. Both excessive and lower expression of the GhLETM1 gene lead to defective stamen development, resulting in shortened filaments and indehiscent anthers with pollen abortion. The results also showed that the phenotype of the shortened filaments was negatively correlated with anther defects in the seesaw model under the ectopic expression of GhLETM1. Moreover, our results notably indicated that the gene requires accurate expression and exhibits a sensitive dose effect for its proper function. This report has important fundamental and practical significance in crop science, and has crucial prospects for genetic engineering of new cytoplasmic male sterility lines and breeding of crop hybrid varieties.