Molecular Cloning And Functional Analysis Of Non-expressor Of Pathogenesis-related Genes1from Gladiolus Hybridus

Gladiolus is one of the famous bulb flowers in the world, which is widely cultivated in China. However their gender degrades seriously due to vegetative propagation for a long time. They suffer from varieties of diseases during the production, resulting in the quality and yield of the bulbs and cutting flowers decreasing. It becomes the main limiting factor on the better quality and more effective production of gladiolus in China. NPR1is not only a key regulator of system acquired resistance (SAR) and induced system resistance (ISR), but also the important regulator of basic resistance and Pto-mediated resistance. Furthermore, NPR1also exert its regulatory role in PR gene expression through interaction with transcription factors TGA. Over-expression of NPR1and TGA2in Arabidopsis can induce downstream genes expression, and then enhance the diseases resistance of plants. The project is planned to clone NPR1and TGA2from Gladiolus hybridus. We investigated their functions through bio-informatics, qRT-PCR, bimolecular fluorescence complementation (BIFC) system, and complement Arabidopsis nprl mutant. The main results are as follows:1) Virus-induced gene silencing in Gladiolus hybridus. The conserved PDS gene sequence was isolated from Gladiolus hybridus. The PDS gene was designated as GhPDS (GenBank accession number: KC344859). Vacuum infiltration of cormels and young plants with the GhPDS-VIGS vector effectively down-regulated the PHYTOENE DESATURASE ortholog GhPDS gene and also resulted in various degrees of photobleaching in Gladiolus hybridus. The variegated plants showed a mean reduction of2.5-fold in GhPDS mRNA transcript levels compared with untreated plants. However, stronger GhPDS suppression results were obtained in almost completely photobleached plants, which exhibited a3.9-fold reduction on average for vacuum infiltration of young plants.2) Isolation of GhNPR1and GhTGA2cDNAs. The cDNAs of GhNPR1and GhTGA2were isolated from Gladiolus hybridus. The GhNPR1gene was designated GhNPR1(GenBank accession number:KJ769203). The full-length GhNPR1, containing a1767bp ORF (open reading frame) region, encodes a protein of588amino acids of a sequence with an estimated molecular weight of65.94kDa and an isoelectric point (pI) of6.36. The GhTGA2sequence was deposited in GenBank (Accession number:KC344858). The ORF of GhTGA2is1296bp in length. The deduced GhTGA2protein had431amino acids and its calculated molecular weight was47.43kDa. Alignment of amino acid sequence of GhTGA2revealed that it is highly similar to previously reported TGA2s.3) Gene expression pattern of GhNPRl. The expression level of GhNPR1in leaves was approximately half the level in cormels and roots, and that the flowers showed the lowest level of expression. Moreover, GhNPR1transcripts were also detected in the leaves after SA treatments. The expression of GhNPR1in leaves peaked12h (about3.8fold) after treatment with1mM SA. However, expression of GhNPR1was not induced after treatment with H2O.4) Interaction of GhNPR1with GhTGA2in transient expression assay. GhNPR1was localized in the nucleus and the cytomembrane of tobacco leaf epidermal cells transiently transformed via Agrobacterium tumefaciens-mediated transformation. GhTGA2-GFP was detected in the nucleus. Tobacco leaf epidermal cells were transiently co-transformed with35S::GhNPRl-YFPN and35S::GhTGA2-YFPc. A strong fluorescence signal was observed in the nuclei.5) Complementation of an Arabidopsis npr1mutant. In order to test whether the GhNPR1gene complements the nprl mutant and plays a role in basal resistance, we tested the resistance of npr1mutants expressing GhNPR1to local bacterial infection with virulent Pseudomonas syringae pv. tomato DC3000(P.s.t DC3000). Growth of P.s.t. DC3000was assayed at0and3dai. The similar bacterial growth was observed in Col-0and npr1/GhNPR1, whereas the well-known enhanced susceptibility mutant nprl harbored an over10-fold higher titer of bacteria than Col-0and npr1/GhNPR1plants.6) Enhanced susceptibility to Curvularia gladioli infection in GhNPRl-silenced plants. Vacuum infiltration of young plants with the GhNPR1-VIGS vector effectively down-regulated the GhNPRl gene. qRT-PCR analysis revealed that the transcript levels of GhNPRl in plants infected with TRV-GhNPR1were approximately70%lower than those in the plants infected with TRV at three weeks post vacuum infiltration. Silencing of GhNPR1resulted in a decrease of disease resistance against Curvularia gladioli. The size of Curvularia gladioli lesions in leaves of the GhNPR1-silenced plants was increased by60%, when compared with those in TRV-infiltrated plants. In conclusion, these results suggest that GhNPRl and GhTGA2play important roles in the SA-dependent systemic acquired resistance in Gladiolus hybridus.