Study On The Function And Mechanism Of The TaSGT1 During Wheat-Puccinia Striiformis F.sp. Tritici Interaction

Wheat stripe rust,caused by Puccinia striiformis f.sp.triici（Pst）,an obligate biotrophic fungus,severely endangers the growth of wheat and threatens the food security of China.Cultivation of resistant cultivars is the most effective and safe approach to control wheat stripe rust.The frequent pathogenicity variation of Pst leads to rapid loss of the resistance of applied cultivars,resulting in the outbreaks of epidemics.Therefore,studying the mechanism of host resistance mediated by resistance-related factors and the pathogenesis of Pst are of great significance for the disease control and the development of strategies for sustainable and effective control of stripe rust.SGT1 plays an important role in plant resistance against pathogen infection,participating in plant broad-spectrum resistance and many resistance（R）gene mediated resistance responses.Therefore,exploring the new mechanism of TaSGT1 participating in the disease resistance,and clarifying how the pathogen effector regulates TaSGT1 during the interaction between Pst and wheat will provide an important theoretical basis for expanding new control strategies of Pst.In this study,two interacting targets of TaSGT1,Pst SIE1 and TaMAB265,were obtained by yeast two-hybrid（Y2H）.The molecular mechanism of Pst SIE1 regulating TaSGT1 during Pst-wheat interaction was systematically studied.Meanwhile,TaMAB265 was preliminarily characterized as a disease-resistance factor.The results are summarized as follows.1.TaSGT1 is an important factor in wheat resistance to Pst.The expression of TaSGT1 was up-regulated in the early stage of Pst infection detected by q RT-PCR.BSMV-VIGS technology was used to silence TaSGT1.The results showed that the disease resistance of the silenced plants to Pst was significantly reduced,as the reduction of reactive oxygen species（ROS）and the increase of the Ratio of fungal biomass were observed during infection.The expression of TaPR1、TaPR2、TaWRKY53 and TaXa21were decreased as similar as in TaSGT1-silenced plants and TaSGT1-TaRAR1 co-silenced plants,suggesting that TaSGT1 is an important factor wheat resistance against stripe rust.2.The effector Pst SIE1 promotes the infection of Pst by targeting TaSGT1 and interfering with the formation of TaSGT1-TaRAR1 complex.In order to understand the mechanism how TaSGT1 is regulated by effectors,the Pst effector Pst SIE1（SGT1 Interacting Effector 1）was screened out by yeast two-hybrid（Y2H）,and the interaction of TaSGT1 and Pst SIE1 was further confirmed in vivo and in vitro through GST Pull-down and Co-Immunoprecipitation（Co-IP）assays.Sequence analysis showed that Pst SIE1 encodes a protein of 208 amino acids with a signal peptide with secretory function.q RT-PCR analysis showed that the expression of Pst SIE1 was up-regulated in the early stage of Pst infection.Transient expression of Pst SIE1 in Nicotiana benthamiana showed that Pst SIE1 inhibits programmed cell death（PCD）induced by Bax,Pst candidate elicitor Pst322,NLPs from Oomycetes,and Vm E02 from Valsa mali.In order to confirm that Pst SIE1 participates in Pst infection,the RNAi and overexpression transgenic wheat materials of Pst SIE1 were created.The resistance of Pst SIE1-RNAi transgenic material to Pst was significantly enhanced,as the growth and development of Pst was significantly inhibited,the colony area and biomass were reduced were observed during infection,and the expression of TaPR1,TaPR2,TaWRKY53 and TaXa21 was increased.The resistance of Pst SIE1 overexpression plants to Pst was significantly weakened,and the accumulation of reactive oxygen species caused by Pst infection was reduced,besides the expression of TaPR1,TaPR2,TaWRKY53 and TaXa21 genes was significantly suppressed.Pst SIE1 and TaRAR1 competitively bind to TaSGT1 through in vivo LUC competition test and in vitro Pull-down competition test.In conclusion,the effector Pst SIE1 promotes the infection of Pst by targeting TaSGT1 and interfering with the formation of TaSGT1-TaRAR1complex.3.A TaSGT1 interacting protein,TaMAB265,was identified to positively regulate the resistance of wheat to Pst.In order to further explore the mechanism of TaGST1 mediated disease resistance,it was found that TaSGT1 might interact with the MATH-BTB protein TaMAB265 through the affinity library of Pst-wheat interaction.The interaction between them was confirmed by Y2H,Co-IP and GST Pull down assays.Sequence analysis showed that TaMAB265 belongs to the plant MAB subfamily,encoding a protein of 347 amino acids,containing the conservative MATH domain and a BTB/POZ domain of the MAB family,and is located on chromosome 7B and 7D of wheat.Y2H assay showed that TaMAB265 interacts with CUL3a of both wheat and Arabidopsis,which confirmed that TaMAB265 participates in CRL^BTB ubiquitination pathway in wheat.q RT-PCR assay showed that TaMAB265 was induced by Pst infection and salicylic acid treatment.Transient silencing of TaMAB265significantly reduced wheat resistance to Pst.And the accumulation of reactive oxygen species and the relative expression of defense related genes TaPR1,TaPR2 and TaPR5 in the TaMAB265-silenced plants were significantly decreased.In conclusion,TaMAB265 is a positive regulator of wheat resistance against Pst.Taken together,this study analyzed the function of TaSGT1 in the interaction between wheat and Pst.We identified a Pst effector Pst SIE1 that interacts with TaSGT1 and analyzed its function.Further we revealed that Pst SIE1 is an important effector that regulates host immunity by targeting TaSGT1 and interfering with the formation of TaSGT1-TaRAR1complex.Moreover,we found that TaSGT1 positively regulates wheat resistance to Pst by targeting TaMAB265.This study provides theoretical foundation for sustainable control of wheat stripe rust.