Colonization of Azorhizobium caulinodans in wheat and nutrient-related miRNA expression

【Objectives】Microbial fertilizer, especially organic materials inoculated with plant growth-promoting bacteria, is becoming a popular type of fertilizers as the environmental concernes caused by over use of chemical ferlitizers, the extension of pollution-free agriculture production. In this paper, wheat seeds were inoculated with Azorhizobium caulinodans ORS571 (A.caulinodans), and the mechanism of nutrient-related miRNAs and colonization of green fluorescence protein (GFP)-labeled A.caulinodans in wheat seedlings were investigated.【Methods】Wheat seeds were infected with A.caulinodans, the root samples of 6 d and leaf samples of 12 d after inoculation were made into slides, scanned using laser confocal microscope. The distribution and colonization of GFP-A.caulinodans in wheat seedlings were detected. Trizol method was used to extract RNA in wheat seedling roots of 6 h, 12 h, 24 h, 48 h, 72 h and 96 h after inoculation. Through the tail adding and reverse transcription reaction, the miRNA within total RNA of samples was synthesized to cDNA. Using -tubulin as reference gene, the expression patterns of miRNAs response to nutrient element were validated by real-time quantitative PCR. The relative expressions of miRNA were calculated with 2-CT method. Using default parameters, the target genes of 6 miRNAs were predicted on psRNATarget online software. 【Results】 1) The laser scanning result shows that GFP-A. caulinodans can colonize in epidermal cells, intercellular spaces of lateral roots, root tip breakages and root hairs. GFP-A.caulinodans can also be detected in root vascular tissue and leaf stoma. 2) The expression of 6 nutrient-related miRNAs varies with inoculation time. The expression of miR164, miR167 and miR827 are upregulated and then down-regulated, and those of miR169 and miR398 are almost consistent. The relative peak expression of miR164, miR167, miR169 and miR398 appear at 12 h after inoculation and the expression are 4-13, 2-84, 2-46 and 3-99 fold of control. The relative peak expression of miR827 occurs at 24 h after inoculation, 2.17 fold of the control. The expression of miR399 shows a trend of down-regulated first and then up-regulated. The relative expression of miR399 reaches the lowest point at 24 h after inoculation, equaling 0.21 fold of the control. 3) Target prediction shows that the target genes of 6 miRNAs code NAC1 transcription factor, Auxin response factor, HAP transcription factor, Cu/Zn-superoxide dismutase, Ubiquitin-conjugating E2 enzyme PHO2 and SPX-MFS Subfamily protein respectively.【Conclusions】GFP-A.caulinodans can colonize in roots of wheat seedlings via root hairs and root tip breakages and ascend into the leaves around stomas. The relative expressions of N, P and microelements related miRNAs can be up-regulated through the inoculation of A.caulinodans. Healthy root morphology is constructed for more efficient uptake of nutrients.