Functional Analyses Of GbWRKY1 From Cotton In Plant Development And Defense Response

Verticillium wilt as the major disease leads to annual yield losses in cotton production. Previous observations and studies demonstrate that there seems to be a close relationship between cotton development and V. dahliae infection. For example, short-season cotton plants are always more susceptible to V. dahliae and the prevalence of cotton Verticillium wilt is most severe after flowering in the field. But the underlying molecular mechanism for such a phenomenon remains unclear. In this study, our genetic and molecular analyses demonstrate multiple functions of GbWRKYl, which was proved to be induced by V. dahliae infection, in the plant defense response and during development. In addition, we also presented a model to explain how GbWRKYl prioritizes development over defense during infection of V. dahliae, which may provide a novel insight into the interplay between cotton and V. dahliae. The main results are as bellow.1. Our study showed that GbWRKY1 was induced by V. dahliae strain V991 at the early stage of infection. Inoculation assays demonstrated that downregulation of GbWRKY1 via VIGS (Virus-induced gene silencing) and RNA interference (RNAi) strategy can increased cotton resistance to Botrytis cinerea and V. dahliae, whereas upregulation of GbWRKY1 attenuated cotton resistance to V. dahliae and B. cinerea. We also generated two GbWPKY1-overexpressing Arabidopsis lines. And similar to the cotton disease resistance assay, overexpression of GbWRKY1 in Arabidopsis also compromised resistance to B. cinerea and V. dahliae. Meanwhile, GbWRKY1-overexpressing plants showed an early-flowering phenotype, whether inoculated with V. dahliae or not. This indicates that GbWRKYl is involved not only in the regulation of plant immunity but also in plant development.2. Gene expression pattern analysis suggested that GbWRKY1 transcript was significantly and rapidly induced by MeJA, while no significant differences in the endogenous level of JA were found between the wild type and GbWRKY1 transgenic cotton lines. In addition, overexpression of GbWRKY1 can negatively affect the JA-mediated defense response and JA-induced anthocyanin accumulation. These results demonstrated that GbWRKY1 might be an important negative regulator in the JA signaling pathway.3. Under normal growth conditions, GbWRKY1-overexpressing cotton and Arabidopsis displayed GA-associated phenotypes, including long petiole and pale green leaves in cotton, and early flowering in Arabidopsis, coupled with the down-regulation of the putative targets of DELLA and a reduced sensitivity to the GA biosynthesis inhibitor paclobutrazol (PAC). Our genetic results further indicated that the early-flowering phenotype from the overexpression of GbWRKY1 depends on the activation of SOC1, which participates in GA-mediated flowering time control. These results demonstrate that overexpresssion of GbWRKYl in cotton and Arabidopsis can promote GA signaling transduction.4. Expression analysis indicated that only AtJAZ1 was up-regulated via the overexpression of GbWRKYl in Arabidopsis, and similar results were also found in GbWRKYl-overexpressing cotton lines. Based on genetic results, we found that GA-related phenotypes of GbWRKYl-overexpressing plants depend on the constitutive activation of JAZ1s. These results indicated that GbWRKY1-orchestrated balance between the JA and GA signaling pathways might be mediated through the interaction of JAZ1-DELLA.5. Promoter analyses showed that there were many W-box (TTGACC) sequences and TGAC core sequences, which were the DNA-binding sites of WRKY transcription factors, found within the 2-kb promoter sequence of AtJAZ1 and GhJAZl. We also show that GbWRKYl is capable of transactivating GhJAZ1 via binding to the TGAC core sequences, but AtJAZl binds via the W-box (TTGACT) and the TGAC core sequence, as revealed by dual-luciferase reporter assays and electrophoretic mobility shift assays.In summary, our data suggested that the early activation of GbWRKY1 after V. dahliae infection might be regulated through a feedback loop of JA signaling pathway and prioritizes development over pathogen defense, but the detailed mechanism needs to be explored further.