Functional Analyses Of Leaf Position And Asparagine Synthetase Gene In Regulation Of Disease Resistance

Plant asparagine synthetases (ASN) are encoded by a small gene family. As one of aminotransferases, ASN uses energy producing from hydrolysis of ATP to catalyze the asparagine synthesis by amidation of aspartate using either glutamine or ammonium as an amino donor. Asparagine is the major form for translocation of organic nitrogen in plants, and thus ASNs are essential to nitrogen metabolism and translocation, which may affect plant disease resistance. Therefore, it is conceivable that ASN may play a role in plant disease resistance. Yet, to date, document in the role of ASN in plant disease resistance is rare. Additionally, analysis of pathogen inoculation is required for evaluation of plant disease resistance. Many factors from pathogen, environment and plant affect the result of pathogen inoculation analysis. Such factors include inoculum concentration and activity, environmental temperature, relative humidity and light, plant age, leaf stage and position. In this study, effect and mechanism of leaf position on disease resistance were studied, and then function of tomato ASN genes in disease resistance was revealed employing virus-induced gene silencing (VIGS) technique and using transgenic plants. Finally, a set of ASN transgenic rice lines were identified. The obtained main results are as follows:(1) Effect of leaf position on plant disease resistance was demonstrated and molecular mechanisms underlying leaf-associated disease resistance were revealed. Through analyzing effect of leaf position on three types of resistance including basal resistance, effector-triggered immunity (ETI) and nonhost resistance of seven pathosystems between three plant hosts and four pathogens, it was made clear that leaf-associated disease resistance exists ubiquitously and its effect is pathosystem-dependent. The anti-fungal basal resistance to Sclerotinia sclerotiorum in Arabidopsis thaliana and Nicotiana tobacum, the anti-bacterial basal resistance to Pseudomonas syringae pv. tabaci (Pstab) in N. benthamiana and ETI conferred by RPS2to P. syringae pv. tomato (Pst) DC3000(AvrRpt2+) in Arabidopsis were stronger in leaves in upper positions (Adult and Reproductive phases) than in those with in lower leaves (Juvenile phase), while Xanthomonas oryzae pv. oryzae (Xoo)-induced nonhost resistance in N. benthamiana and Arabidopsis and Pst DC3000-induced nonhost resistance in N. benthamiana showed just the opposite. Microarray expression profiling assays demonstrated that hundreds of genes, which are involved in defense response, phytohormone biosynthesis and signaling, cell wall composition and reinforcement and calcium signaling, were differentially expressed among the leaves of different phases. Mutants sidl and sid2-3, which are involved in salicylic acid (SA) transport and biosynthesis respectively; ein2, which is involved in ethylene signaling;jar1-1, which is a component of jasmonic acid (JA) signaling; and aba1and aao3, which are involved in abscisic acid (ABA) biosynthesis, didn’t display significantly different resistance to S. sclerotiorum, mutants ein2, ein3-1, eto2-1, which are involved in ethylene sensing and signaling, and mutant sidl showed opposite trend of severity of RPS2/AvrRpt2+-conferred HR to wild type plants, while mutant sid2-3exhibited opposite trend of severity of Xoo-induced HR to wild type plants. Collectively, our results unveil that generally phytohormones such as SA, ethylene, JA and ABA are essential to diverse types of leaf position-associated resistance. However, the involved phytohormones are pathosystem-dependent. These results also indicate that leaves at the same positions should be used for inoculation analysis to evaluate plant disease resistance.(2) The ASN gene families in tomato (Solanum lycopersicum) and Nicotiana benthamiana were identified in genome-wide level, and function of the two SIASN genes in resistance was revealed employing Tobacco rattle virus (TRV)-based VIGS technique. Bioinformatics analyses showed that tomato and N. benthamiana ASN families comprised2and6genes respectively, which were classified into two groups. Silencing of SIASN1in tomato reduced resistance to Pst DC3000and S. sclerotiorum, however enhanced resistance to Pythium aphanidermatum and Cf-4/Avr4-dependent hypersensitive response (HR), while did not affect resistance to TRV and Zoo-induced HR. Whereas silencing of SIASN2in tomato reduced resistance to TRV, Pst DC3000and S. sclerotiorum, however enhanced resistance to P. aphanidermatum, while did not alter Cf-4/Avr4-dependent and Xoo-induced HR. This result demonstrates that both SIASN genes positively regulate tomato resistance to Pst DC3000and S. sclerotiorum while negatively regulate resistance to P. aphanidermatum. However, they differ in regulating some other resistance such as resistance to TRV and Cf-4/Avr4-dependent HR. In addition, silencing of SIASN1did not change expression of SIASN2. However, silencing of SIASN2increased expression of SIASN1by4.1folds. This indicates that SlASN2may execute inhibitory effect on SlASNI expression.(3) Transgenic N. tabacum lines overexpressing SIASN2(SIASN2-OE) were generated and function of SIASN2in resistance was revealed using T2SIASN2-OE plants. Three lines of SIASN2-OE transgenic tobacco were produced. Two of them exhibited significantly phenotypes such as curled and abnormal leaves, necrosis in newly developed leaves, water-sunken necrosis in stem and died finally. However, the remaining SIASN2-OE line grew normally as wild type plants. Results of qRT-PCR expression analysis demonstrated that SIASN2expression in all three lines was significantly higher than in wild type, but no significant difference exhibited among the three plants. T2plants of the SlASN2-OE transgenic line were used for functional analysis. The results showed that the transgenic plants increased the resistance to S. sclerotiorum, Pstab, Tobacco mosaic virus (TMV) and Cucumber mosaic virus (CMV), but reduced the resistance to P. aphanidermatum, while did not affect the resistance to Meloidogyne incongnita and the Cf-4/Avr4-dependent and Xoo-induced HR. It indicates that SIASN2positively regulates resistance to S. sclerotiorum, Pstab, TMV and CMV but negatively regulates resistance to P. aphanidermatum. These results are consistent with those obtained using VIGS in tomato. Additionally, SlASN2-OE transgenic plants enhanced tolerance to0.4M NaCl stress, indicating that SIASN2may also positively regulate plant salt tolerance.(4) T4OsASN overexpression and RNAi transgenic rice lines generated previously in our lab were subjected to identification for true transgenic plants by β-glucuronidase (GUS) staining analysis, kanamycin resistance check and growth and development phenotype observation. The obtained true transgenic plants lay a good foundation for functional analyses of the OsASN genes. Results of GUS staining analysis and kanamycin resistance check showed that5lines including OsASN4-3、 OsASN4-4、OsASN4-5、OsASN4-7and OsASN4-13were positive lines but were still not homozygous. Fifty-six homozygous transgenic rice plants were identified. Observation of growth and development phenotype revealed that plants of OsASN3-OE lines showed retarded growth and development, reflected as delayed heading compared with wild type plants. The percentage of full grains among total grains per head in OsASN3-RNAi rice was lower than50%, which was significantly lower than in wild type (68%). Collectively, OsASN3may play an essential role in regulating rice growth and development.