Validation And Functional Analyses Of Genes Coding Agglutinin And SWI/SNF-related Chromatin Binding Proteins In Pinellia And Arabidopsis

In their natural environments, plants are continuously threatened by fungal, bacterial, viral pathogens and insects. This results in enormous reduction of yield and quality in many crop plants. Compared to chemical prevention, exploring the efficacy of genes encoding anti-fungal proteins is considered to be an effective and safe method to control fungal pathogens. Consequentially, it is important genes encoding novel anti-fungal genes be explored for deployment in agriculture. Endophytic fungi can produce a vast array of secondary metabolites some of which are similar to those produced by their host plants. Pinellia ternata agglutinin genes and two secondary metabolite syntheses genes were chosen as the target genes to screen all the isolated strains from Pinellia ternate, and the strains with antifungal activities were used as microbial pesticide in biological control. This work also can provide the scientific basis for solving the shortage of Pinellia resource.Vascular wilt is an important disease caused by the fungal pathogen Fusarium oxysporum. F.oxysporum is difficult to eradicate from agricultural soils. This pathogen is soil-borne and can stay dormant in the soil until the environment is suitable to germinate. The model plant Arabidopsis thaliana is also infected by this pathogen and therefore it provides a suitable model to investigate the mechanism of resistance （or susceptibility） with an expectation that the results would provide increased knowledge on plants defense against F.oxysporum.In the study, I studied potential roles of the Pinellia agglutinin genes and a SWI/SNF-related chromatin binding protein gene from Arabidopsis for their roles in providing resistance against F. oxysporum. The main results were listed as follows:1. The full length of genomic Pinellia agglutinin genes, including Pinellia ternata agglutinin （PTA）, P. pedatisecta agglutinin （PPA）, P. cordata agglutinin （PCA）, P. integrifolia agglutinin （PIA） and P. peltata agglutinin （DYA）, were cloned and analyzed. The sequence comparisons showed Pinellia agglutinin genes had high similarity to each other, and this gene family contained two conservative B-lectin domains and three mannose binding sites. Bacterial expression showed that the protein weight of Pinellia agglutinin proteins are between 27 kDa and 28 kDa, and the efficient expression of Pinellia agglutinin protein could be achieved after induction with 0.8 mmol·L-1 IPTG in E. coil BL21 （DE3） for 2 h at 37"C. Solubility analysis showed that the fused protein mainly existed as inclusion bodies. To investigate the function of the three mannose binding sites in Pinellia ternata agglutinin, the three mutant Pinellia ternata agglutinin genes （named MBS1, MBS2, and MBS3, respectively） were cloned by overlap extension PCR, respectively. Bacterial expression of the encoding proteins showed that the protein weight of MBSs agglutinin proteins is about 27 kDa, and the solubility analysis showed that the fused protein mainly existed as inclusion bodies.2. The plant expression vector of Pinellia agglutinin and MBSs agglutinin were constructed and transiently expressed in N. benthamiana. The subcellular location results showed that the PTA was uniformly localized in cell membrane, while the PPA was located in the plasma membrane as spots, the rest of three Pinellia agglutinins were non-uniformly localized to the plasma membrane. The results also showed the subcellular location of Pinellia ternata agglutinin was altered when the mannose binding sites were mutated, the MBS1, MBS2 and MBS3 agglutinin were found in the ER （endoplasmic reticulum） rings around nuclei, as well as in the cell membrane. The leaves of N. benthamiana were syringe infiltrated with Pinellia agglutinin, MBSs agglutinin and pI1300-35S-eYFP, and assayed for ROS 36 h after injection. The flg22 was used as an elicitor. The results showed that ROS generation was induced both in the leaves of N. benthamiana transiently expressed Pinellia agglutinin and pI1300-35S-eYFP, but their production were quite different. The leaves of N. benthamiana expressing PTA showed the highest levels of ROS production in response to flg22, followed by PPA and PCA, the leaves of N. benthamiana expressing PIA showed the lowest level of ROS production that was even lower that the control treatment. The results also showed the ROS production was significantly decreased in the leaves of N. benthamiana expressing MBS1 when compared with MBS2, MBS3 and PTA, respectively. The ROS production did not show significant difference in the leaves of N. benthamiana expressing MBS2, MBS3 or PTA.3. Eighty-one endophytic fungi were isolated from the leaves, stems and bulbs of Pinellia ternata by tissue inoculation culture. Among them,40 strains were isolated from leaves,17 from stems and 24 from bulbs. According to internal transcribed spacer sequences （ITS）, all isolated strains were represented 11 orders,14 families and 18 genera. Cladosporium and Penicillium were the dominant genera, while ZH15 and ZH77 may be two new species. Three gene, encoding Pinellia ternata agglutinin （PTA）, nonribosomal peptide synthetases （NRPS） and polyketide synthase （PKS）, were used as the marker genes to screen all the isolated strains. The presence of PKS and NRPS were detected in 36 and 11 isolated strains respectively, and the PTA gene was amplified in some isolated strains. Six plant pathogens, including Fusarium oxysporum f.sp. niveum, Fusarium graminearum, Pellicularia sasakii, Colletotrichum gloeosporioides, Magnaporthe oryzae and Botrytis cinerea, were used to test the anti-fungal activities of collected endophytic fungi by agar plate. Thirty-four isolated strains had antifungal activity against one or more pathogens. The Arthrinium sp. strain ZH13 and ZH35 had broad-spectrum anti-fungal activity and its fermentation liquid can inhibit the growth the hyphe of F. graminearum and B. cinerea.4. The SWI-SNF full-length genomic DNA sequence and its promoter （+3000bp upstream of the gene） were cloned and analyzed, the result showed the sequence contained some cis-acting regulatory elements and four characteristic domains. To explore the possibility of the SWI-SNF involved in Arabidopsis, the gene expression pattern was investigated by qRT-PCR. SWI/SNF was expressed in roots, shoots and flowers with a low expression level. No obvious differences in the expression level of SWI-SNF were found in these tissues.5. We isolated two homozygous T-DNA insertion lines designated as swisnf1 and swisnf2 for this gene and we also generated the SWI-SNF over-expression transgenic plants and the complement transgenic plants. The swi/snf mutants showed increased resistance to F. oxysporum, while the disease phenotype was quite opposite in over-expression transgenic lines. In addition, the complement transgenic plants displayed indistinguishable disease phenotype from that of wild-type plants, indicating that SWI-SNF played a necessary role in plant resistance to F. oxysporum.6. To determine whether the SWI-SNF is responsive to MeJA, the transcription of SWI-SNF was monitored in Col-0 after MeJA treatment via different time courses. The results showed that SWI-SNF gene was induced by MeJA. To investigate whether some JA signaling marker genes, like COI1, MYC2 and LBD20 affects the expression of SWI-SNF during MeJA treatment, the SWI-SNF expression level was investigated in coil, myc2 and lbd20 mutants. The results suggested the plant defense response mediated by SWI-SNF is COI1-, MYC2- and LBD20- dependent. To determine whether SWI-SNF play a role in JA signal pathway, some marker genes involved in JA signaling pathway was monitored in Col-0 and swisnf mutants, the transcription level of genes, including JAZ5, JAZ10, VSP2, Thi2.1 and PDF1.2, were significantly induced in the swisnf mutants when compared to Col-0.7. To determine whether the SWI-SNF is responsive to SA, the transcription level of SWI-SNF was monitored in Col-0 after SA treatment via different time courses. The results showed that SWI-SNF gene was induced by SA. To investigate whether the SA regulatory gene NPR1 has a role in the induction of SWI-SNF by SA, the SWI-SNF expression level was investigated using nprl-2 mutants and Col-0. The results suggested the plant defense response mediated by SWI-SNF is NPR1- dependent. To determine whether SWI-SNF play a role in SA signal pathway, some marker genes involved in SA signaling pathway was monitored in Col-0 and in swisnf mutant. PRI, PR2 and PR5 were significantly induced in the swisnf mutants when compared to Col-0.8. To determine whether SWI-SNF is responsive to Foxysporum, some marker genes involved in JA and SA signaling pathways was investigated in Col-0 and the swisnf mutants by qRT-PCR after inoculations. The results showed that transcript levels of some genes, including JAZ5, JAZ10, VSP2, Thi2.1, PR2 and PR5, were significantly induced in the swisnf mutants when compared to Col-0. While transcript levels of other genes, like PDF1.2, PR3, PR4 and PR1 did not show significant differences compared to Col-0.9. The other SWI/SNF-related chromatin binding protein named SWI-SNF1 （Tair accession number Atlg20290）, which shared 51% amino acid sequence similarity with SWI-SNF, was approximately -10000bp downstream of the SWI-SNF. To determine whether SWI-SNF1 is also involved in Arabidopsis against Foxysporum, we identified one homozygous T-DNA insertion mutant swisnf3 （Salk₀12847C）, we found that the swisnf3 mutant also shows resistance to F.oxysporum, but the SWI-SNF1 gene is not response to SA or MeJA treatment. To determine whether the SWI-SNF and SWI-SNF1 are functionally redundant, we generated swisnfl swisnf3 double mutants. The swisnf1 swisnf3 double mutant showed similar disease phenotypes to the swisnf single mutant.