Functional Characterization Of Three VQ Motif-containing Genes In Rice-Xoo Interaction

Plants are frequently exposed to various environmental stresses,including abiotic and biotic stresses such as drought,freezing,nutrient deficiency and pathogen infection and therefore have developed complex mechanisms to quickly respond and finally adapt to these stresses and that direct to immune responses.These responses are usually referred to as disease resistance.Rice(Oryza sativa L)is the staple food crop for more than half of the world's population.Rice bacterial blight,caused by the bacterial pathogen Xanthomonas oryzae pv.oryzae(Xoo)is the most devastating bacterial disease of rice,causing tremendous yield loss each year all over the world.Studying the underlying mechanism of rice-pathogen interaction will not only provide genetic resources for rice breeding but also advance our knowledge towards plant-pathogen interactions.The VQ motif-containing proteins(designated as VQ)are a class of plant-specific proteins with a conserved and single short FxxxVQxLTG amino acid sequence motif.Recent studies revealed that,VQ proteins in Arabidopsis have shown to influence to diverse physiological processes.Expression analysis of rice VQ genes under several stress treatments were assessed,specifically,following infection with the Xoo,treatment with abscisic acid(ABA),or exposure to drought.However,the functions of rice VQ genes are unknown.In this study,the function of OsVQ11(LOC_Os03g20440),OsVQ13(LOC_Os03g47280)and Oplant sVQ40(LOC_Os03g09045)in response to Xoo inoculation with rice were characterized.We used overexpression and artificial microRNA(amiRNA)approach to generate transgenic rice lines in Zhonghua 11,a japonica cultivar.In this study we found that OsVQ11-overexpression(VQ11-oe)and OsVQ13-overexpression(VQ13-oe)plants with Zhonghua 11 background showed increased resistance to Xoo strain PX0347,and the enhanced resistance was associated with expression level of OsVQ11 and OsVQ13.Two VQ13-oe T1 families showed correlation between disease area and expression at significant level.We also generated VQ11-ami,VQ13-ami and VQ40-ami transgenic lines to suppress the function of these genes using artificial microRNA(amiRNA)approach.We found that suppressing VQ11 and VQ40 genes do not show any correlation between disease area and expression level for the VQ11-ami and VQ40-ami To and T1 generations.Two T1 transgenic families of VQ13-ami(VQ13-ami32 and VQ13-ami36)showed enhanced susceptibility to Xoo and correlation was found between their disease area and expression at significant level.Mitogen-activated protein kinase(MAPK)cascades play important roles in various defense signal transduction pathways.OsMAPK5 is a negative regulator of disease resistance,and its suppression can enhance rice resistance to Xoo.As a repressor,OsMPK6 negatively regulates systemic acquired resistance(SAR)in response to Xoo infection.Suppressing or knocking out of OsMPK6 enhanced rice resistance to different races of Xoo.It also functions as an activator that regulates local resistance to Xoo.As a plant specific transcription factor family,WRKY transcription factors also involve in rice-pathogen interactions.MAPKs can phosphorylate its downstream components whereas WRKY proteins act as transcription factor with various types of proteins related to disease resistance.In order to know whether the OsVQ genes(OsVQ11,-13,-30)have interactions with MAPK and WRKY proteins,we performed yeast two hybrid(Y2H)assay.We found that,these three VQ genes interacted with MPK5 and MPK6 in yeast cell.However,OsVQ11,-13,-30 showed no interaction with a set of group I WRKY proteins.Based on our results,we expect,VQ genes analyzed in this study may have potential role in rice-Xoo interactions,which still need more experimentation for further confirmation.Taken together with our data,it indicates that these results of OsVQ proteins will provide useful resources in future functional studies of OsVQ genes.