Functional Analysis Of An LRR Domain-haboring Potential Type ? Effector In Xanthomonas Oryzae Pv.Oryzae

Xanthomonas oryzae pv.oryzae?Xoo?,the causal agent of bacteria leaf blight,is a Gram negative bacterium,it enters into the plants via wounds and hydathode,and then multiply in the vessel xylem.Xoo infects a narrow range of host,especially rice and wildrice stem.In some serious cases the disease could be devastative for rice production.The rice-Xoo interaction has been considered as an important model in phytopathology.Previous researches revealed that type? effectors and type ? secretion system through which type ? effectors are secreted into host cells play important roles in the pathogenesis.In the Philippine Xoo strain PX099A the potential type ? effector PXO₀3420,which is also referred as HpaF,was found structurally similar with the type ? effector PopC from Ralstonia.Domain prediction by SMART?Simple Modular Architecture Research Tool?revealed three leucine rich domains?LRR?in HpaF.LRR domain has been proven to involve protein-protein interactions,and play important roles in the functioning of some type ? effectors.In this study,the mutant affected in hpaF showed significantly reduced mobility?46.62%less?and virulence?51.81%less?while provoke delayed non-host hypersensitive reaction on Ricinus communis l.,and these phenotypes can be complemented by pLAFR3 harboring the open reading frame of PXO₀3420,indicating that HpaF might involve in the mobility and is required for the full virulence of PXO99A.The phylogenic analysis on PXO₀3420 was carried out by MEGA5 and the results revealed that most of the genes that are closely related to PXO₀3420 have been proved to be type ? effectors.To identify the interacting protein of HpaF in the rice cultivar Nipponbare by yeast two-hybrid system,a cDNA library of Nipponbare has been created by using the classical switching mechanism at 5' end of RNA transcript?SMARTTM?method.The conversion rate and the exogenous fragment recombinant rate of the library attained 2.57×106 pfu/mL and 87.6%,respectively,indicate that the quality of the library is enough for further yeast two-hybrid analysis and the newly construct AH109/pGBKT7₀3420 was used as bait.26 positive clones were identified from the yeast two-hybrid analysis and were verified by quantifying the?-galactosidase activity.The sequencing of the potential interacting clones revealed that proteins containing WHy and PRK domain might interact with HpaF in PX099A.