Characteristic analysis of powdery mildew resistance of ubiquitin ligases VpUIRP2 and VpUIRP3 from Chinese wild Vitis pseudoreticulata

Abstract: 【Objective】We cloned ubiquitin ligases VpUIRP2 and VpUIRP3 from highly resistant acces-sion of Chinese wild V. pseudoreticulata‘Baihe-35-1'and studied their expression characteristics in order to provide available genes for improving disease-resistance of European grape (Vitis vinifera L.). Thegenes of VpUIRP2 and VpUIRP3 were introduced into European grape, and the disease resistance of trans-genic plantlets against grapevine powdery mildew (Uncinula necator) was analyzed.【Methods】The patho-gen-related genes were screened in a PM-inoculated c DNA library of‘Baihe-35-1'. Among the ETSsidentified, two ETSs were predicted to encode a putative RING finger protein, belonging to the E3 ligasefamily. Then homologous cloning was employed to obtain coding sequences of VpUIRP2 and VpUIRP3.The bioinformatics methods were used to analyze conversed domains, chromosome localization informa-tion and deductive protein properties. In order to study genes' expression pattern, pathogen inoculationand phytohormone treatment were conducted. Uncinula necator were inoculated on the leaves of‘Baihe-35-1'and‘Cabernet Sauvignon'(V. vinifera‘Cabernet Sauvignon')by friction extrusion method, whileleaves treated with dd H2 O were used as control. Each sample was randomly collected with three leaves inthe positions of third or fourth knot from the shoot tip at 0, 6, 12, 24, 36, 48, 60 and 72 h post inoculation.Phytohormone treatment was conducted as follows: 1 mmol·L^-1 salicylic acid(SA) solution was sprayed onthe leaves of‘Baihe-35-1'and‘Cabernet Sauvignon'and the samples were collected at 0, 1, 3, 6, 9, 12,24 and 36 h post treatment. Genes' responses to Uncinula necator and salicylic acid were identified viaq RT-PCR method. Besides, their subcellular localizations were showed by PEG-mediated transformationof Arabidopsis protoplasts. The coding sequences(CDSs) of VpUIRP2 and VpUIRP3 were cloned intop BI221-GFP to construct the subcellular localization vectors, and subcellular localizations of two ubiqui-tin ligases were gained through PEG-mediated transformation and fluorescence observation. In order tofurther explore the function of VpUIRP2 and VpUIRP3, these two genes were transformed into V. vinifera‘Thompson Seedless' and V. vinifera‘Red Globe'grape plants via Agrobacterium-mediated transforma-tion. The putative transgenic lines were detected sequentially by resistance screening, PCR test and South-ern blot analysis. A series of statistical approaches and observations were conducted as follows to evaluatetransgenic grapevine resistant to powdery mildew. The inoculation of powdery mildew was made on thethird and fourth leaves from the shoot tip of transgenic and wild plants of‘Thompson Seedless' and‘RedGlobe'. Phenotypes difference between transgenic and wild plants were recorded on 12 days post inocula-tion. During the treatment, leaves were collected at 24, 48 and 168 h for staining and counting numbers ofspores and hyphae with microscope. Aniline blue staining method was used, blood counting chamber wasused to analyze Uncinula necator growth condition for evaluating disease resistance of transgenic plants.【Results】Two RING-type ubiquitin ligase genes VpUIRP2 and VpUIRP3 were obtained, and their codingsequences were submitted to Gen Bank(http://www.ncbi.nlm.nih.gov/Web Sub/?tool= genbank) and two ac-cession numbers were provided(KU519335, KU519336). With bioinformatics analysis, we found thatubiquitin ligase UIRP2 was located on grape chromosome 5 and UIRP3 was located on grape chromosome8. VpUIRP2 coding sequences contained 954 bp, encoding 317 amino acids with a C3H2C3 type RINGFinger conserved domain in the middle. In contrast, VpUIRP3 coding sequences contained 1 641 bp, en-coding 546 amino acids with a C3H2C3 type RING Finger conserved domain in the C-terminal. Expres-sion of VpUIRP2 and VpUIRP3 were induced by Uncinula necator inoculation and SA treatment.VpUIRP2 was localized in the cytoplasm while VpUIRP3 was localized in the whole cell. These results ofsubcellular localization of VpUIRP2 and VpUIRP3 were consistent with domain analysis. VpUIRP2 con-tained a transmembrane domain, it might be localize in the membrane of cytoplasm. VpUIRP2 and VpUIRP3 genes were introduced into‘Red Globe'and‘Thompson Seedless' by genetic transformationvia Agrobacterium. 13 transformed lines of‘Red Globe'with VpUIRP2 gene and 2 transformed lines of‘Red Globe'with VpUIRP3 gene were obtained. And 2 transformed lines of‘Thompson Seedless' with VpUIRP2 gene and 2 transformed lines of‘Thompson Seedless' with VpUIRP3 gene were obtained.Grape transformed lines were further tested by PCR and Southern blot. Results showed that 33.3% of thekanamycin-resisted plantlets with VpUIRP2 were transgenic lines. These data indicated that VpUIRP2 was introduced to‘Red globe'and‘Thompson Seedless' while the target bands of VpUIRP3 transformation strains were not detected. Transgenic and non-transformed lines were assayed for evaluating the resis-tance to powdery mildew (U. necator). The hyphae growing on the leaves of transgenic plants at 168 hpiwere lower than those of the controls. Furthermore, the number of conidiophores counted at 168 hpi by ani-line blue staining and statistics analyzation showed that the difference between the VpUIRP2 overexpres-sors and the non-transformed control was statistically significant. These data clearly demonstrated thatoverexpression of VpUIRP2 contributed to a reduction in the susceptibility of the transformed V. vinifera plants to U. necator.【Conclusion】Ubiquitin ligase genes VpUIRP2 and VpUIRP3 from Chinese wild V.pseudoreticulata‘Baihe-35-1'could be involved in the resistance to grape powdery mildew. Additional-ly,the genes VpUIRP2 and VpUIRP3 could be used as disease-resistant genes for improving the resis-tance of European grape variety.