Screening And Functional Analysis Of AVR-PikH4 Rice Proteins Of Rice Blast Fungus Acinetobacter

Rice blast caused by Magnaporthe oryzae infestation,which is an important limiting factor in rice production.It has the characteristics of rapid propagation,wide range of occurrence,and serious damage,which seriously threatens rice production.It is currently the most economical and effective method to control rice blast by aggregating multiple major disease resistance genes to breed new rice varieties.Due to the lack of understanding of the specific mechanism of resistance genes in rice and the diversity and variability of the physiological races of rice blast fungus,the resistance of the varieties cultivated by simple aggregation of several disease-resistance genes is not obvious or will be promoted in the field.Quickly loses resistance,leading to the recurrence of rice blast again.To study the role of avirulence genes of rice blast fungus in rice is helpful to elucidate the pathogenic mechanism of avirulence genes and reveal the possible mechanism of plant resistance resistance,laying an important theoretical foundation for the cultivation of new rice varieties with long-lasting broad spectrum and high resistance to rice blast.In this study,yeast two-hybrid system was used to identify and analyze the interaction proteins of the non-toxic protein AVR-PikH4 of Magnaporthe grisea.The results are as follows:1.The leaves of different infestation stages(1 d,2 d,3 d,and 4 d)after the inoculation of rice blast fungus GD0193 with high resistance to blast rice(H4)were obtained by space mutagenesis in the early stage of the research group.The combination of chain-specific nuclease(DSN)homogenization technology and SMART(switching mechanism at 5' end of RNA transcript)database construction method successfully established the homogenization of rice leaf blades infected with rice blast fungus.The yeast two-hybrid cDNA library has a high capacity(2.2×10 6cfu/mL)and an insertion length of more than 400 bp.The homogenization effect is significant,which lays the foundation for the cost,time and screening of the library.2.According to gene-gene theory,H4 is highly resistant to the rice blast representative strain GD0193 with a broad pathogenic spectrum in Guangdong,and the avirulence gene AVR-PikH4 corresponding to the resistant gene Pik-H4 is assumed to be present in strain GD0193,in order to further clarify whether GD0193 is The presence of AVR-PikH4 and analysis of its genetic relationship with the allele AVR-Piks.We cloned AVR-PikH4 from the rice blast fungus GD0193.The results indicate that the gene is identical to the reported AVR-Pikh.3.In order to further understand the pathogenic pathways involved in the avirulence gene AVR-PikH4 in rice,we successfully constructed the bait plasmid pGBKT7-AVRPikH4?SP(full-length AVR-PikH4 has self-activating activity).The decoy plasmid does not have self-activation activity.And cytotoxicity.Further yeast two-hybrid screening results using a homogenized cDNA library of H4 showed that 45 rice proteins interacting with AVR-PikH4 were obtained in primary screening using SD/-Trp/-Leu/-His/-Ade/+X-24-interacting proteins were confirmed by rescreening on ?-Gal medium plates.Further,PCR and sequencing were used to remove interaction and frame-shift mutation interactions.Finally,15 candidate interaction clones were obtained.Five candidate interacting proteins that may be related to pathogenicity were picked from them,and their full-length cDNAs were cloned.The pGADT7 plasmid was verified by two-hybridization with pGBKT7-AVRPikH4?SP.The results show that there is a direct interaction between AVR-PikH4 and LOC_Os06g28590.1,LOC_Os02g46030 and LOC_Os06g22960.