The Function Of Atdhyprpl In Systemic Acquired Resistance And Its Characteristics In Subcellular Localization

AtDHyPRPl (.DOUBLE HYBRID PROLINE-RICH PROTEIN1, AT4G22470) is an Arabidopsis gene without intron and its open reading frame is1128bp in length. The protein of38.79kDa in size encoded by AtDHyPRP1contains375amino acid residues and a N-terminal signal peptide composed by23amino acids. Analysis of the amino acid sequence through TMHMM2.0Server indicated that AtDHyPRP1has no transmembrane domain. Similar to the members of lipid transfer protein/protease inhibitor/seed storage protein family, alignment search using the protein-blast online tool in NCBI showed that AtDHyPRP1owned two conservative eight cysteine motifs (8CMs). Owing to the coexistence of two proline-rich domains with8CMs, AtDHyPRP1also is categorized into HyPRP (hybrid pro line-rich protein) family.In the present work, the resistance function of AtDHyPRPl to microbial pathogens and its characteristics in subcellular localization were investigated with overexpression lines (OE), RNA interference lines (RNAi), AtDHyPRPl-GFP transgenic lines of Arabidopsis in Ws genetic background, and with AtDHyPRPl transgenic tobacco lines. Laser scanning cofocal microscopy revealed that AtDHyPRP1-GFP fusion proteins were localized to cell surface and was mainly distributed in phloem and xylem of vascular tissues. In addition, the stability of AtDHyPRP1-GFP in transgenic Arabidopsis plants could be influenced by infection of Pseudomonas syringea. The green fluorescence in roots will centralized irregularly and will be degraded in leaves after treatment with Pst DC3000.In expression analyses, mRNA was isolated from roots, stems and leaves of wild-type Arabidopsis plants. Quantitative RT-PCR (RT-qPCR) exhibited that remarkable differences in transcription of AtDHyPRP1could be found in different tissues, with a higher mRNA abundance in leaves and a lower mRNA abundance in stems. Further analyses after treatments of the wild-type Arabidopsis plants with phytohormones indicated that AtDHyPRP1could be induced by salicylic acid (SA) and methyl jasmonate (MeJA), and the inductive effect of SA to AtDHyPRPl was higher than that of MeJA. Moreover, both virulent Pst DC3000and avirulent Pst avrRPM1could stimulate the transcription of AtDHyPRP1, and Pst DC3000was more effective than Pst avrRPMl. In nprl-1mutant that the endogenous synthesis of SA is unaffected, the expression of AtDHyPRP1was obviously lower than that of the wild-type plants. All these results suggested that AtDHyPRPl was a downstream mediator after NPR1in S A signal pathway.To identify the function of AtDHyPRPl in SAR (systemic acquired resistance), lower (local) leaves were inoculated with avirulent P. syringae, then the distal leaves were inoculated with virulent P. syringae after several days. The results showed that the RNAi lines of AtDHyPRP1had a weakened SAR and were more susceptible to the bacterial pathogen. In contrast, OE lines exhibited enhanced SAR. RT-qPCR to major genes associated with SAR revealed that the expression levels of AtPRl and AtGST6was decreased in RNAi lines of AtDHyPRPl in comparison with wild-type plants, whereas the expression of these two genes in OE lines of AtDHyPRP1was upregulated. These results confirmed that AtDHyPRP1was involved in defense response of Arabidopsis against pathogens.Besides, the resistance of AtDHyPRP1to fungal pathogens were determined by inoculation with the conidia of Gibberella kujikuroi, and the results showed that the RNAi lines of AtDHyPRPl were more sensitive than the wild-type plants, whereas the infection symptoms of OE lines were alleviated in a certain extent. In AtDHyPRP1transgenic tobacco lines, the major genes of SAR also was upregulated compared to wild-type tobacco plants, the size of lesion spots formed in leaves after infection by G. kujikuroi was decreased, and the disease symptoms were alleviated significantly.