The Molecular Mechanism Of A Rice Bowman-Birk Trypsin Inhibitor Mediating The Interaction Between Rice And Magnaporthe Oryzae

Rice(Oryzae sativa)is one of the major staple crops,providing food for nearly half the world’s population.Rice blast,caused by Magnaporthe oryzae is a global fungal disease of rice,which occurs in different countries and regions.Rice blast management in the past demonstrated that the use of broad-spectrum disease resistant varieties is one of the safest,effective and environmental friendly strategies to control the disease.However,due to the genetic variability and complexity of M.oryzae populations in the field,new resistant varieties may gradually become susceptible in several years.Therefore,research on the molecular mechanism between the rice and M.oryzae interaction will provide guidelines and strategies for developing new disease resistant varieties.In our previous study,we cloned the broad-spectrum resistance(R)gene Piz-t in rice,and our collaborators cloned its cognate avirulence gene AvrPiz-t in M.oryzae.Because Piz-t did not directly interact with AvrPiz-t,we used the yeast two-hybrid system to screen rice host proteins and obtain 12 Avr Piz-t-interacting proteins in rice,named APIP1-12.Among of them,APIP4 encodes a Bowman-Birk type trypsin inhibitor.In this study,we characterized the function and molecular mechanism of the Bowman-Birk trypsin inhibitor APIP4during the interaction between the M.oryzae effector AvrPiz-t and the rice NLR protein Piz-t.First,we performed GST pull-down and co-immunoprecipitation assay to demonstrate the specific interaction between AvrPiz-t and APIP4.The biochemical activity experiments showed that APIP4exhibits trypsin inhibitor activity in vivo and in vitro,while the point mutation APIP4~K198A198A partially loses trypsin inhibitor activity.When Avr Piz-t was coexpressed with APIP4,AvrPiz-t could specifically suppress the APIP4 trypsin inhibitor activity.To study the biological function of APIP4,we created APIP4 knock-out and overexpression transgenic plants.The inoculation assay indicated that knockout APIP4 reduces the resistance to M.oryzae,while overexpress APIP4 enhances the resistance to M.oryzae.Therefore,APIP4 is a positive regulatory factor in regulating rice disease resistance to blast fungus.Notably,the overexpression of APIP4 in rice plants also showed trypsin inhibitor activity.We also found that transgenic plants of AvrPiz-t could inhibit the APIP4 trypsin inhibitor activity.Meanwhile,we also found that Piz-t-HA transgenic plants could enhance the APIP4 trypsin inhibitor activity.Moreover,Piz-t can enhance the APIP4 transcriptional level and protein accumulation.GST pull-down,LUC and Co-IP experiments proved that Piz-t could interact with APIP4.Furthermore,we knocked out APIP4 in Piz-t-HA background and performed the punch inoculation,which indicates that APIP4 knockout plants could not affect the Piz-t resistance against the avirulent isolate,but weakened the resistance to the virulent isolate.Therefore,these results revealed a new mechanism by which the effector protein Avr Piz-t and the NLR protein Piz-t could modulate the rice resistance through antagonistic regulation of the trypsin inhibitor.