?.map-based Cloning And Molecular Characterization Of An Immune-enhanced Lesion Resembling Disease Mutant Lrd6-6 In Rice ?.functional Characterization Of The Chloroplast Targeted DnaJ Chaperonin OsDjA 7/8 In Rice

Rice plants encounter various diseases including blast disease and bacterial blight which cause a lot of loss in production.It's important to breed disease resistance cultivars to guarantee food security.Here we reported the identification of a lesion resembling disease mutant from tissue cultured rice Kitaake and named it lrd6-6.We cloned the gene Lrd6-6 and resolved molecular mechanism of cell death and immune response regulation mediated by the AAA ATPase LRD6-6 and proposed a novel strategy for applying the gene to rice disease resistant breeding procedure.The main results are summarized as below:1.We characterized the lrd6-6 mutant phenotypes and confirmed that the lesion mimic phenotype of lrd6-6 was resulted from accumulated cell death.DAB staining revealed that H2O2 was deposited in lrd6-6.2.Disease resistance measurement showed that the the mutant lrd6-6 conferred resistance to blast caused by Magnaporthe grisea and bacterial blight caused by Xanthomonas oryzae pv oryzae,respectively.3.By combining the map-based cloning method and whole genomic re-sequencing approach,we detected a 1446 bp nucleic acids insertion,in the gene LOC_Os06g03940.We then verified the function of this gene by RNAi and complement test and proved Os06g03940 is the gene Lrd6-6.4.Lrd6-6 was found to express in all these tissues with predominance in leaves.The punctuated signals of LRD6-6 N or C terminal fused fluorescence were colocalized with signals of RabF1/RAR6 fusion protein,a MVBs peripheral membrane protein,further confirmed that the LRD6-6 protein was located in MVBs.LRD6-6 belongs to the AAA ATPase family,and it homologs were reported to function in formation of MVBs and MVBs-mediated vesicular Trafficking.We identified three conserved amino acids,K261,E315 and R372 in LRD6-6 based on alignment with its homologs and constructed the corresponding expression vectors.5.To characterize the features of LRD6-6 protein,we expressed and purified the proteins LRD6-6,LRD6-6K261A,LRD6-6E315Q and LRD6-6R372E respectively and subjected to malachite green based colorimetric reaction to determinate the ATPase activity of these proteins.We found the LRD6-6 was a functional ATPase that able to hydrolysis ATP in vitro while the ATPase activity was abolished for the mutation versions,LRD6-6K261A,LRD6-6E315Q and LRD6-6R372E,respectively,indicating that these amino acid residues,K261,E315,and R372 are all required for the ATPase activity for LRD6-6.Yeast two-hybrid and BiFC analyses revealed that LRD6-6 and the mutation versions,LRD6-6K261A and LRD6-6E315Q,were all able to form homodimers in yeast and N.benthamiana while the LRD6-6R373E not.Transgenic expression of Lrd6-6 carrying either of the site mutations,LRD6-6K261A,LRD6-6E315Q and LRD6-6R372E,respectively,was not able to restore the phenotype of the lrd6-6 mutant to wild type suggesting that all these three amino acid residues are essential for the biologic role of As all the three residues,are required for ATPase acitivity while the residue E315 is required for homo-dimerazation of LRD6-6,these results also suggest that both ATPase activity and homo-dimerazation of LRD6-6 are essential for LRD6-6 to function biologically in rice.6.Based on previous reports,we presumed that LRD6-6E315Q might play a dominant-negative role for LRD6-6 in rice.To test this hypothesis,we tested the interaction ability of LRD6-6 with the mutation version LRD6-6E3 5Q and revealed their interaction in both yeast and plant.We then overexpressed Lrd6-6E315Q in wild type Kitaake.Interestingly,we found that the transgene plants expression the mutation protein,LRD6-6E315Q exhibited lesion spots phenotype like the mutant lrd6-6.These results showed that the LRD6-6E315Q was able to compromise LRD6-6 function by forming a functionally inactive homo-dimer,then rendering LRD6-6 dominant-negative.These results also suggest that the mutant gene Lrd6-6E315Q could be utilized as a gene trap to suppress the inhibitory regulation of the ATPase LRD6-6 in immunity and thus providing a way to use Lrd6-6 for rice disease resistance breeding.7.To identify downstream components of the immunity and cell death resulting from disruption of the LRD6-6 ATPase in the lrd6-6 mutant,we performed a genome-wide transcript comparative analysis on the lrd6-6 mutant and Kitaake using the RNA-seq approach.We discovered 1223 genes that differentially expressed between Kitaake and lrd6-6 mutant.Among them,980 were up-regulated while 243 were down-regulated in lrd6-6 compared to Kitaake.GO analysis mainly classified these differentially expressed genes(DEGs)into cellular components that predicted to participate in MVBs formation and its mediated vesicular trafficking.The expression of the genes was verified by qRT-PCR.The carboxypeptidase Y from Arabidopsis was fused with GFP and used as a marker to detect the MVBs mediated vesicular trafficking and we demonstrated that this process was compromised in Lrd6-6 dysfunction cells.8.Pathway analysis found that the DEGs were mainly enriched in three pathways,phenylpropanoid biosynthesis(ko00940),diterpenoid biosynthesis(ko00904)and phenylalanine,tyrosine and tryptophan biosynthesis(ko00400)which responsible for synthesis of antimicrobial metabolites,lignin,phytoalexins and serotonin,respectively.Consistently,we detected more lignin and phytoalexins accumulated in the mutant lrd6-6,the Lrd6-6E315Q overexpression plant while reverted to wild type level in the transgenic complement plant.9.To further dissect the molecular mechanism of LRD6-6 in regulation of immunity and cell death,we performed yeast two hybrid screen using LRD6-6 as a bait.Y2H screened a LRD6-6 interactor,the ESCRT-? component SNF7 homolog OsSNF7.Bimolecular fluorescence complementation(BiFC)further confirmed the interaction between them.By using CRISPR/Cas9 approach,we obtained three independent lines of transgenic plants with OsSnf7 knocked out.Unfortunately,none of the OsSnf7 knocked out plants exhibited obvious lesion spots compared with the wild type rice Kitaake.Further analysis revealed at least two other SNF7 homologs in rice may function redundantly with OsSNF7.Thus,to well elucidate the roles of SNF7s in immunity and cell death in rice it would require to develop double or even triple knock-out mutants for Snf7 homologous genes in future studies.We also identified the VPS2 homolog OsVPS2 was also able to interact with LRD6-6,further supporting our previous conclusions.Chloroplast is an important organelles that converting light energy into chemical energy and releasing oxygen which is essential for humans.DnaJ proteins belong to chaperones of Hsp40 family that ubiquitously participate in various cellular processes.Previous reports have shown that the molecular chaperone DnaJs play essential roles in various plant organelles and participate in various pathways in regulation plant growth and development.In chloroplast,DnaJs have been reported to regulate the development and function of the chloroplast and even the responses of the plant to both abiotic and biotic stresses.However,little is known about the roles of DnaJs in rice.In this study,we reported the identification and functional characterization of the DnaJ encoded by LOC_Os05g26902/LOC_Os05g26902(OsDjA7/OsDjA8)in rice.OsDjA7/8 is a type I DnaJ protein.We found that the gene OsDjA7/8 was expressed in all collected tissues,with a priority in the vigorous growth leaf.Subcellular localization revealed that the protein OsDjA7/8 was mainly distributed in chloroplast.Reduced expression of OsDjA7/8 in rice led to albino lethal at the seedling stage.Transmission electron microscopy observation showed that the chloroplast structures were abnormally developed in the plants silenced for OsDjA7/8.In addition,the transcriptional expression of the genes tightly associated with the development of chloroplast was deeply reduced in the plants silenced for OsDjA7/8.Collectively,our study reveals that OsDjA7/8 encodes a chloroplast-localized protein and is essential for chloroplast development and differentiation in rice.