Screening For Proteins Interacting With Plant Pathogenic Oomycete RXLR Effector AVR1B

Oomycetes include Phytophthora, downy mildews and Pythium, most of which are well-known plant pathogens and cause devastating damages to many food and ornamental crops worldwide. Phytophthora sojae is an important model species for understanding genetics, biology and pathology of oomycetes. Phytophthora root rot of soybean caused by P. sojae is a destructive disease, which causes a loss of more than 2 billion dollars in the worldwide annually. Investigation on the interaction between oomycete pathogens and their host plants and the disease resistance mechanisms at the molecular level will facilitate the development of novel disease control strategies.Pathogen effectors are known as Avr factors when they are recognized by plants. Many effectors encoded by the oomycete pathogens contain a conserved RXLR-dEER motif in the downstream sequence of their signal peptides, they are also called RXLR effectors. RXLR effectors play an important role in the process of pathogenesis. P. sojae Avr1b plays not only an important role as an avirulence gene on the soybeans containing cognate resistance gene Rps1b, but also a virulent role in P. sojae infection in the absence of Rps1b.In this study, a cDNA library, constructed using germinated cysts of P. parasitica and diseased leaf tissues of Arabidopsis thaliana infected with P. parasitica, were screened for proteins interacting with the Avr1b effector protein using a yeast two-hybrid approach. The bait protein gene mAvr1b was amplified by PCR and inserted into the bait vector pGBKT7 to generate recombinant plasmid named pGBKT7-mAvr1b for yeast-two-hybrid screening.A total of 12 unique candidate proteins were obtained by screening 2×10~7 cDNA clones and were confirmed by cotransformation to be Avr1b-interacting. Sequence alignment showed 6 homologous genes in Arabidopsis thaliana. Four of them were chosen for further analysis and 2 were confirmed to be interacting with Avr1b effector. This study facilitats further investigation on translocation of Avr1b effector and its virulence mechanisms in host plant cells.