Subcellular Localization And Resistance To Botrytis Cinerea And Pseudomonas Syringae Of A Similar Lipid Transfer Protein AtDHyPRP1from Arabidopsis Thaliana

The protein encoded by AT4G22470of Arabidopsis thaliana consists of a signal peptide, two proline-rich domains (PRD) and two eight cysteine motifs (8CM). Owning to no report about the function of AT4G22470, it has been designated as AtDHyPRP1(DOUBLE HYBRID PROLINE-RICH PROTEIN1) in this work. Alignment of the deduced amino acid sequences by Clustal W showed that the8CM of AtDHyPRP1shares high similarity with plant lipid transfer proteins in the positions of eight cysteine residues. The theoretical isoelectric point (PI) and the molecular weight of AtDHyPRP1were8.68and38789.6Da, respectively. Its chemical formula is C1772H2800N444O487S21and the number of total atoms is5524. In addition, there is a23-amino-acid-long signal peptide in N-terminus of AtDHyPRPl, and the possibility is0.999. AtDHyPRP1has no transmembrane domain and the average hydrophobicity value of amino acid residues is1.103, indicating this protein is a hydrophobic protein. Prediction of the second structure suggested that the N-terminus of AtDHyPRP1contains helix, coil and sheet. The microarray results in database showed that AtDHyPRP1can be induced strongly by infection of Pseudomonas syringae pv. tomato DC3000.In order to understand the function of this protein, the encoding sequence was amplified by PCR and inserted into the downstream of the constitutive CaMV35S promoter to produce a plant binary expression vector pRI101-AN-AtDHyPRPl. Transgenic tobacco plants were regenerated by leaf disc method and were confirmed by PCR, Southern blotting, RT-PCR and Northern blotting. Inoculation of the detached leaves with the conidial suspension of Botrytis cinerea revealed that the expression of AtDHyPRP1in tobacco plants could improve the fungus resistance remarkably, and the infection sites on transgenic tobacco leaves accumulated large amounts of H2O2. Trypan blue staining showed that a large number of cells in pathogen infected leaves of wild-type qinyan95plants were died in comparison with the leaves of the transgenic tobacco plants transformed with AtDHyPRP1, indicating AtDHyPRP1 plays an important role in defense process of Arabidopsis against biotic stresses. At the same time, the coding sequence of AtDHyPRP1amplified by PCR from Ws ecotype was used to construct the fusion expression vector pCAMBIA1302-AtDHyPRP1-GFP and three lines of35S::AtDHyPRP1-GFP transformed Arabidopsis plants were produced by floral dip method. Observation under laser scanning confocal microscope showed that AtDHyPRP1was localized to cell surface, in consistent with the results of bioinformatics and resistance analyses. It suggested that AtDHyPRP1might play special function after secretion to outside of the cell and was involved in plant defense system against pathogens.In order to further clarify the mechanism of the resistance of AtDHyPRP1in plant defense system against pathogens, four overexpressing lines (OX), six RNA interference lines (RNAi) of Arabidopsis were produced by floral dip method, and five homozygous mutant lines were screened by PCR using the primer pairs of AtDHyPRP1and Bar from the progeny of T-DNA insertion lines. RT-PCR analyses showed that the expression of AtDHyPRP1was upregulated in overexpressing lines and decreased remarkably in RNA interference lines in comparison with the wild-type plants, and no transcript of AtDHyPRP1could be detected in homozygous mutant lines. In wild-type Ws plants (Wt), AtDHyPRP1could be induced by virulent Pst DC3000and avirulent Pst avrRpm1, and the expression of it peaked at24h after Pst DC3000and8h after Pst avrRpm1inoculation respectively, followed by slight decrease, and increased again after at48h. Four days after inoculation, the order of the infection degree of different materials by B. cinerea is OX<Wt<RNAi<KO and the order of the propagation speed of the virulent Pseudomonas syringae pv. tomato DC3000in different materials also is OX<Wt <RNAi<KO. Observation under laser scanning confocal microscope indicated that infection of35S::AtDHyPRP1-GFP transgenic Arabidopsis plants with virulent Pst DC3000lead to obvious changes of the localization of AtDHyPRPl, including irregular distribution and disordered arrangement. In SAR analyses, systemic leaves were challenged with virulent Pst DC3000four days after inoculation of one or two local leaves with avirulent Pst avrRpm1(AV) or MgSO4(MV). Compared to MV treatment, the chlorosis and necrosis phenotypes of Wt, OX and RNAi plants were significantly alleviated in AV treatment, and most of the leaves remains green. In contrast, the phenotypes of T-DNA homozygous plants had no significant difference after AV and MV treatment, and most of the leaves were chlorotic. Statistical analysis indicated that the virulent Pst DC3000propagated much faster in systemic leaves of Pst avrRpm1-induced (AV) homozygous mutant plants than in equivalent leaves of wild-type plants, while the growth of the virulent Pst DC3000had no significant difference in leaves of uninduced (MV) homozygous mutant and wild-type plants four days post inoculation. All these results suggested that AtDHyPRP1plays a vital role in defense system of Arabidopsis against pathogen infection.