Cotton Cytochrome P450 CYP82D Regulates Systemic Cell Death

Verticillium dahliae is a phytopathogenic fungus that causes serious soil-borne vascular disease in cotton, which is referred to as the’cancer’of cotton. However, sea island cotton often show more resistance to V. dahliae compared to upland cotton, therefore there may have to be some high resistance cotton germplasms in them. In our previous work, we isolated an significant different EST (Expressed Sequence Tag) from a SSH (Suppression Subtractive Hybridization) library in a screen for genes involved in cotton disease resistance following inoculation with Verticillium dahliae. Three highly similar genes (above 94%) were identified in G. hirsutum genotype YZ1 each with 1,569-nucleotide ORFs (Open Reading Frames) and putatively encoded proteins of 522 amino acids, with conserved domains that are characteristic of eukaryotic P450 proteins. Sequence analysis revealed that they share 55% identity with PtCYP82D2 but only 48% with AtCYP82C2. Thus, They are novel P450s in cotton, named as CYP82D1, CYP82D2 and CYP82D3.To explore the role of this P450 subfamily in cotton, we knocked down the expression of the gene or gene family using RNA interference (RNAi) technology for the 3’-UTR regions or conserved regions respectively. Meanwhile we construct the overexpression vectors of GhCYP82Dl and GhCYP82D2. We transform these genes or gene fragments to upland cotton line YZ1 by agrobacterium-mediated genetic transformation. Most GhCYP82D RNAi seedlings showed lesion mimics phenotype on the stems and most died without any pathogen infection, consistent with the level of gene downregulation. So the gene family was named SILENCING-INDUCED STEM NECROSIS (SSN), and the three members GhCYP82Dl, GhCYP82D2 and GhCYP82D3 were named SSN1, SSN2 and SSN3, respectively. No obvious phenotypic differences were observed between the 3’-UTR specifical silenced plants and WT seedlings. Crossing was employed using different specific 3’-UTR silenced lines to examine the functional redundancy among the gene members.The results showed that silencing any two members of the SSN family could induce the lesion phenotype. Thus, SSN members may have functional redundancy.To determine the function of SSN in the cotton immunity system, Illumina sequencing was employed to identify genes that were differentially expressed in roots between the SSN-RNAi and WT seedlings. A large number of genes have been shown to directly participate in JA synthesis and signalling pathways. The expression patterns for genes involved in the JA signal pathway were examined in seedlings after experimental wounding. The results show that JA-related genes expression levels and jasmonate contents were more rapidly induced in SSN-RNAi than WT seedlings. However, JA-related genes transcripts and jasmonate contents were apparently attenuated in OE lines after a wound treatment compared with WT seedlings. However, JA biosynthesis and signalling never could induce lesion mimics phenotype. Jasmonates are kinds of oxylipin derived from octadecanoid pathway. To assess the metabolic differences between the SSN-RNAi and OE lines, fatty acid levels were determined. Interestingly, only LA levels were significantly reduced in the OE seedlings compared with WT and SSN-RNAi. At the same time the cotyledon oxylipins were also measured, and 9-HOD/T,13-HOD/T and 9/13-KOD dramatically accumulated in the OE seedlings. These data indicate that SSN is involved in the octadecanoid pathway. SSN likely acts on C18 fatty acids and competes for substrates with LOXs. Also, HR-like phenotype of SSN-RNAi plants is related to imbalance in LOX pathway.In addition, the SSN-mediaed oxylin pathway plays an important role in systemic cell death and defence induction. But the specific compound or signal is unclear now, we hypothesize that it may be have some relationship with the mobile signal required for systemic acquired resistance. The unknown oxylipin-derived compound or signal may be a important component in systemic acquired resistance.We also propose that JA and the systemic cell death play a key role in cotton V. dahliae resistance. The combination of these two parts disease-resistant mechanism makes SSN-RNAi plants show the resistance to Verticillium wilt. SSN acts as a valve to regulate defence response on pathogen infection. The plant controls the ’on/off’ signal for disease resistance via modulating SSN metabolic pathway.