Functional Studies On ERF49 Gene In Plant Response To Verticillium Wilt And Saline-alkali Stress

Plants are confronted with various environmental constraints during growing,such as Verticillium wilt and soil saline-alkali stress,which cause huge economic losses to agricultural production.AP2/ERF（APELATA 2/Ethylene responsive factor）family transcription factors play important roles in plant response to stress.AtERF49,one member of the AP2/ERF family,was cloned in our previous study,whcih showed that AtERF49 gene is involved in plant response to stress such as salt,but its molecular mechanism is still unclear.In order to further explore the function ofERF49 gene in plant response to Verticillium wilt and saline-alkali stress,the Arabidopsis wild-type Col-0,the transgenic Arabidopsis overexpressing AtERF49,and the CRISPR/Cas9 mutant erf49 were analyzed in this study under treatment with Verticillium dahliae LX2-1 and 150 mmol/L saline-alkali solution（Na HCO₃and Na₂CO₃molar ratio 9:1 mixed）,respectively.We also observed the phenotype of the cotton line GhERF49 silenced by VIGS（virus-induced gene silencing）after inoculation with Verticillium Wilt strains LX2-1 for 2 weeks.q RT-PCR analysis was conducted to detect the the expression levels of stress response genes RAB18 and RD29A and lignin synthesis-related genes;The cell death of leaves was visualized by trypan blue staining and the chlorophyll fluorescence parameters of Arabidopsis leaves were determined by chlorophyll fluorescence-imaging-gas exchange simultaneous measurement system.The main results are as follows:1.The results of stem longitudinal section observation and V.dahliae recovery experiments indicated that virus-induced gene silencing of GhERF49 resulted in higher resistance of cotton plant to V.dahliae LX2-1 than the control.Further anaysis of q RT-PCR revealed that the expression levels of lignin synthesis-related genes Gh CCo AOMT1,Gh C4H1,Gh F5H1 and Gh CAD6 were increased significantly in GhERF49-scilencing cotton.2.The overexpression of AtERF49 resulted in higher susceptibility of plants to V.dahliae,while the mutant erf49 showed higher resistance to V.dahliae.Trypan blue staining analysis showed that the number of dead cells in leaves of AtERF49-overexpressing plants was more than that of erf49 mutant.Further anaysis of q RT-PCR showed that the expression levels of At PAL4 and At CCo AOMT1 genes were decreased in AtERF49-overexpressing plants after inoculation with V.dahliae,while increased in erf49 mutants.Under saline-alkali stress,the leaves of the AtERF49-overexpressing plants turned slightly yellow,while the leaves of erf49 mutant seriouly wilted and bleached.q RT-PCR results showed that overexpression of AtERF49 gene could up-regulate the expression of RAB18 and RD29A genes.Chlorophyll fluorescence parameters measurement results showed that the Y（II）and（q P）in AtERF49-overexpressing plants were significantly higher than those of Col-0,and（NO）and（q N）were significantly reduced.However,the opposite trend was apparent in erf49 mutant.In addition,overexpression of AtERF49up-regulated the expression of the photosynthetic responsive gene rbc L and down-regulated the expression of psa A.Together,these research results indicate thatERF49 gene may play a negative regulatory function in plant resistance to Verticillium wilt by affecting the expression of lignin biosynthesis-related genes;Under saline-alkali stress.AtERF49 gene might be involved in plant resistance to saline-alkali stress by regulating the expression of stress-responsive genes and the efficiency of photosynthesis.However,the functional differences of AtERF49 gene in plant responses to Verticillium wilt and saline-alkali stress require further in-depth study.