Molecular Mechanism Of Interaction Between Arabidopsis Thaliana And Oxalic Acid

Many physiologically and ecologically different fungi can secrete oxalic acid (OA), which plays a crucial role during pathogen infection process. OA-producing pathogens, such as S. sclerotiorum and Botrytis cinerea, can infect hundreds of plants and cause severe loss of crops, but the material resistant to the fungi is limited due to the extensive host range. So, there is a great need to study the defense response to OA-producing pathogen and seek the resistant materials. Arabidopsis thaliana possesses good characteristics, such as small genome, simple structure, small figure, short life period, high propagating rate and self-pollination etc. Moreover, its genomic sequence was completed on the end of 2000, which provided high homology between Arabidopsis and higher plants on the genome. So, Arabidopsis has been used as a model plant for the analysis of plant defense response. In this paper, the interaction between Arabidopsis and OA was studied in three aspects: first, OA-resistant mutants were obtained from screening a chemical-inducible mutant library and their molecular mechanism was studied. The ultimate objective is to isolate the OA-insensitive gene and obtain the S. sclerotiorum resistant material, thus, decrease the use of pesticide and improve the quality of environment and security of foods; second, Oxalic acid is catabolized by two major pathways, i.e. decarboxylation and oxidation. So, oxalate decarboxylase was cloned from Bacillus subtilis 168, and oxalate oxidase was cloned from barley, and their plant expression vectors were constructed and transgenic plants were obtained by floral dip. The S. sclerotiorum resistance was detected among the transgenic plants; third, oligo microarray and microRNA microarray were used to investigate the differentially expressed genes and miRNAs under the stress of OA, thus further understand the mechanism of pathological effects of OA and provide the theoretical foundation of biological control on OA-producing pathogens.The major results in this study are as follows:1. Based on the effects of different concentration OA treatments on the growth of Arabidopsis ecotype Columbia-0 seeds, the threshold concentration of OA was obtained. The screen system was to sow the sterilized seeds of mutant library on agar plates of MS medium (pH 6.5, calcium free) supplemented with OA (final concentration 1.2 mmol/L), estradiol (final concentration 10μmol/L), and MES (final concentration 1g/L).2. Putative OA-insensitive mutants were screened from the mutant library constructed by the laboratory of Professor Zuo Jiruo from the Chinese Academy of Sciences (about 6000 individual lines) using MS medium containing 1.2 mmol/L OA and 10μmol/L estradiol. About 300 putative OA insensitive mutants were obtained. Those mutants were again screened on the selective medium and five mutants, D33, D74, D154, D282, and D630 with enhanced resistance toward OA were obtained. Through TAIL-PCR, the flanking sequences were rescued. The sequences were blasted against TAIR database. The result indicated that the T-DNA of mutant D33 was inserted between At2g39720 (zinc finger) and At2g39730 (Rubisco activase) (GenBank accession No. EF591991), and the T-DNA junctions of the other four mutants were the same, all inserted in the same site of the first intron of At5g10450 (14-3-3 protein GF14 lambda) (GenBank accession No. EF591992).3. The expression of At2g39690,At2g39700, At2g39720 and At2g39750 were up-regulated in mutant D33 compared with wild type by semi-quantitation RT-PCR.4. The over-expression vectors of At2g39690,At2g39700 and At2g39720 were constructed and the transgenic plants were obtained by floral tip, and the resistance to S. sclerotiorum and OA will be detected among the transgenic plants.5. The putative OA-insensitive mutants were inoculated with agar plugs of S. sclerotiorum using detached leaves or in planta, mutants were more resistant than wild type. So the method to screen OA-resistant mutant, thus obtain S. sclerotiorum resistant mutant is practicable. It is possible to isolate the OA-resistant gene and obtain S. sclerotiorum resistant material.6. Oxalate decarboxylase (Yvrk) was cloned from Bacillus subtilis168, and oxalate oxidase was cloned from barley, and their plant expression vectors were constructed and transgenic plants were obtained by floral dip. The transgenic plants possess resistance to S. sclerotiorum. This result indicates a novel approach to develop transgenic plants resistant to fungal infection.7. Oligo microarray was used to investigate the differentially expressed genes under the stress of OA. The bioinformatics analysis indicated that OA induces the jasmonic/ethylene signaling, not salicylic acid signaling. The expressions of phenylpropanoid pathway, WRKY transcription family, cytochrome P450 family, ATP-binding cassette transporter family and heat shock family proteins were up- regulated, which indicating these pathway or family proteins were involved in the plant response to OA.8. MicroRNA microarray was used to investigate the differentially expressed micro-RNAs under the stress of OA. Three differentially expressed microRNAs and 9 predicted_miRNAs were obtained. Among them, one down-regulated microRNA is Ppt-miR1211 from Physcomitrella patens, and its target mRNA is At3g14520- terpene synthase based on miRU, another down-regulated microRNA is Ptc-miR3911 from Populus trichocarpa, which is identical to Ath-miR399b, and its target mRNA is At2g33770- a ubiquitin-conjugating E2 enzyme, both the two targets up-regulated in oligo-microarray. The only up-regulated differentially expressed microRNA is Ath-miR858, whose target mRNAs are At2g47460(DNA transcription factor),At3g08500(DNA transcription factor)and At3g20310(ethylene responsive element- binding family protein. All the three target mRNAs were down-regulated in oligo-microarray, indicating the negatively-regulating function of microRNA.