Verticillium wilt, caused by soil-borne fungus Verticillium dahliae, is one of the most destructive diseases in cotton. An LRR-TM gene we cloned from Gossypium barbadense (GbVe) significantly improved Verticillium wilt resistance of transgenic Arabidopsis. However, the resistance mechanism of the GbVe is still obscure. In the present study, we identified that the GbVe located in Dt09, homologous to tomato Ve1 but possessing lower identity with other reported cotton Ve genes in At01 and /or Dt01, and further investigated the molecular mechanism of the gene in resistance against V. dahliae. Ectopic expression of GbVe activated salicylic acid signaling, elevating the expression levels of EDS1, NDR1, PR4 and PR5 in transgenic Arobidopsis plants, and enhanced the accumulation of lignin, which improved resistance to V. dahliae. These results provide us important clue to further investigate the molecular mechanism of GbVe in cotton itself. Overexpression of GbVe in cotton (G. hirsutum) made defense response to Verticillium wilt enhanced. Transgenic cotton led to activation of SA signaling pathway (including cotton ICS1, EDS1, NPR1, NDR1, PR1, PR4 and PR5 genes), causing SA acumulation and lignin increase that confer resistance to V. dahliae. We also detected that the GbVe could interact with NHL13 gene involved in SA signal pathway via a yeast-two-hybrid screening. In the condition of V. dahliae free, GbVe and NHL13 interacted with each other. However, once stressed by the pathogen, the expression of NHL13 gene decreased whereas the expression level of GbVe displayed significantly increased, indicating that the interactions dissociation between GbVe and NHL13. The enhanced GbVe expression further activated defensive genes expression in SA signal pathway as well as regulating H2O2 and lignin content, which improved the Verticillium wilt resistance of cotton. When silenced GbVe gene in cotton, the plants showed more susceptible. Whereas, the plants displayed no obvious disease resistance difference between NHL13-silent and non-silent seedlings. These funding provides insight into molecular mechanism by which plants integrate SA signal regulated by GbVe to protect themselves from V. dahliae infection.

Plants evolve effective mechanisms to protect themselves against multiple stresses and employ jasmonates (JA) as vital defense signals to defend against pathogen infection. Accumulation of JA induced by signals from biotic and abiotic stresses, results in degradation of Jasmonate-ZIM-Domain (JAZ) proteins, and then de-repressed the JAZ-repressed transcription factors (such as MYC2) to activate defense responses and developmental processes. Here, we characterized a JAZ family protein, GhJAZ2, from cotton (Gossypium hirsutum) which was induced by methyl jasmonate (MeJA) and inoculation of Verticillium dahliae. Over-expression of GhJAZ2 in cotton impairs the sensitivity to JA, decreases the expression level of JA-response genes (GhPDF1.2 and GhVSP) and enhances more susceptibility to V. dahliae and insect herbivory. Yeast two-hybrid and BiFC assays showed that GhJAZ2 may be involved in regulation of cotton disease resistance by interacting with more disease response proteins, like pathogenesis-related protein GhPR10, dirigent-like protein GhD2, NBS-LRR disease resistant protein GhR1, and a basic helix-loop-helix transcription factor bHLH171. Unlike MYC2, over-expression of bHLH171 in cotton activates the JA synthesis and signaling pathway, and improves plant tolerance to fungus V. dahliae. Molecular and genetic evidences showed that GhJAZ2 could interact with bHLH171 and inhibit its transcriptional activity, as a result, restrain the JA-mediated defense response. Recent studies have revealed that the SUPPRESSOR OF BIR1-1 (SOBIR1) can interact with multiple receptor-like proteins (RLPs) and is required for resistance against fungal pathogens. We also find that GbSOBIR1 gene could be induced by Verticillium dahliae inoculation. Knock-down of GbSOBIR1 resulted in attenuated resistance of cotton plants to V. dahliae, while heterologous overexpression of GbSOBIR1 in Arabidopsis improves the resistance. We also found that the kinase region of GbSOBIR1 could interact with bHLH171 and contributes to the resistance of cotton against V. dahliae. And the transcriptional activity of bHLH171 is significantly improved when co-expressed with GbSOBIR1 in tobacco. To identify the GbSOBIR1-mediated phosphorylation site of bHLH171, a spectrometric analysis was performed, and phosphorylation defective forms of the bHLH171 protein with serine to alanine mutations were assayed. The results showed that phosphorylation at Ser413 is essential for the physiological function of bHLH171. Collectively, these results demonstrate that multiple signal pathways are employed by cotton during cotton resistance to V. dahliae.

DNA methylation regulates gene expression without changing the original DNA sequence, which regulates a range of functions in plant development and stress responses, maintenance genomic stability, stress response, and among others, but a role in male sterility under HT remains undetermined. In our previous studies, male sterility under high temperature is a critical factor contributing to yield loss in cotton. Using genome-wide total DNA methylation rate measurement by HPLC at three anther developmental stages under normal and high temperature (HT) conditions, we found that the total DNA methylation level of H05 was always lower than that in 84021 under HT, which was in accordance with more differentially expressed genes in H05 than in 84021 under HT (Min et al., 2014). To better understand how DNA methylation addresses HT stress during male reproductive stages, we performed whole genome bisulfite sequencing. Global disruption of DNA methylation, especially CHH methylation (where H=A, C or T), was found in an HT-sensitive line. Changes of 24-nucleotide small interference RNAs were significantly associated with DNA methylation levels. Experimental suppression of DNA methylation led to pollen sterility in the HT-sensitive line under NT, but did not affect the normal dehiscence of anther wall. Further transcriptome analysis of the anther showed that the expression of genes in sugar and reactive oxygen species (ROS) metabolic pathways were modulated significantly, but auxin biosynthesis and signaling pathways were slightly changed, indicating that HT disorders sugar and ROS metabolism via disrupting DNA methylation, leading to microspores sterility. This study opens up a path to create HT-tolerant cultivars using epigenetic solutions.

Yield enhancement is the ultimate goal of crop improvement through genome characterization and manipulation which can be achieved by purposeful genome editing of plant species through alteration of the genetic make by direct gene transformation. Efforts have been made to control insect pests through genetic modification of local cotton varieties through introduction of codon optimized Cry1Ac and Cry2a genes under the control of CAMV35s promoter. Almost 100% mortality of Lepidopteron insects was achieved after 3rd day of detached cotton leaf bioassay when evaluated in lab and showed significant control of these insects in field conditions as well. Similarly, codon optimized cp4 EPSPS transformed in insect-resistant local cotton varieties has shown significant level of glyphosate tolerance after spray at the rate of 1900ml per acre as compared to non-transgenic cotton plants and all grasses which start decaying on 3rd day and showed complete death after 5th day of glyphosate spray assay. Similarly, introduction of different transcription factors, namely WLIM5 in combination with HOX3 and fibre related genes like SUS, AcsA+B, Actin, Aco3 along with pigment structural genes like F3'5'H and DFR flavonoids in different combinations in both Gossypium hirsutum and Gossypium arboreum have resulted in increase of fibre length from 26 mm to 31.5 mm and 18 mm to 26.5 mm respectively also micronaire value was improved from 4.0 to 3.2 and 7.9 to 6.3. Application of molecular breeding and advanced technology like CRISPR CAS 9 system to further enhance the capabilities of the product will be helpful in utilization of full potential of the product developed.