Identification Of Co-Regulatory Components In Responses To Xanthomonas Oryzae Pv. Oryzae Infection And Nitrogen Deficiency Stresses In Rice

Bacterial leaf blight of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is more severe atexcessive nitrogen use in the field. This observation suggested that crosstalk between disease resistanceand nitrogen (N) utilization pathways might exist in rice. However, the overlapping transcriptionalresponses in rice to pathogen and N stresses and co-regulatory components involved in this crosstalkhave not yet been elucidated.In this study, we analyzed the biomass and defense response to Xoo in rice (Oryza sativa L. cv.Nipponbare) at different N conditions. Compared with normal N supply of3mM KNO3, N deficiencysupply of0.3mM KNO3significantly reduced the total biomass. When inoculated with Xoo, rice plantsgrown in N deficiency condition displayed weaker disease symptoms and much shorter lesion lengthsthan those in normal N supply condition. Bacterial numbers in the rice leaves grown in N deficiencycondition also decreased. These results indicated that disease resistance to Xoo was enhanced in rice atN deficiency condition. Therefore, we speculated that there might be co-regulatory components inresponding to Xoo infection and N deficiency condition in rice.Thus, experiments were designed and carried out to reveal the regulatory mechanisms and co-regulatorycomponents at transcriptional and post-transcriptional levels in this crosstalk. The results are listedbelow.We searched for differentially-expressed miRNAs in rice at Xoo infection and nitrogen deficiencycondition by high-throughput sequencing, and found61miRNAs only responded to Xoo stress,80miRNAs only responded to low nirtogten stress. Interestingly,72miRNAs responded to both stresses,28out of which were chosen for confirmation by qRT-PCR experiments. Expression of miR5076,miR5072, miR156and miR1320were induced, whereas miR812, miR169, miR396, and miR2869wererepressed at both stresses. Comparatively, expression of miR398and miR399were induced at Xoostress, but repressed at low nitrogen stress, and the expression of miR5540was repressed at Xoo stressbut induced at low nitrogen stress. By analyzing the promoter sequence of these miRNAs, severalknown stress-responsive elements were identified, such as defense and stress-responsive element(TC-rich repeats), defense and wounding-responsive element (W box), and so on. We also identifiedsome target genes of the co-regulated miRNA. The roles of the miRNAs and their target genes will befurther studied.Digital gene expression analysis (DGE) based on Solexa/Illumina sequencing was used to investigatetranscriptomic responses to Xoo inoculation, low N treatment, or a combination of both stresses.248differentially-expressed genes (DEGs) of rice in overlapping responses to both stresses were identified,which were involved in innate immunity, nitrogen metabolism, oxidation-reduction, proteinphosphorylation and signaling. Expressions of40randomly-selected DEGs were validated byquantitative real-time PCR (qRT-PCR) assays. Temporal expression of six genes selected from different functional categories assayed by qRT-PCR suggested that N condition was the dominant factor whenboth stresses were present.In conclusion, some miRNAs and DEGs were identified by high-throughput sequencing and qRT-PCR,which were very likely to be involved in the molecular crosstalk between Xoo stress and low nitrogenstress in rice. The findings will help us to understand the co-regulatory mechanisms of rice responses tomultiple biotic and abiotic stresses. Putative co-regulatory components identified here will be furtherfunctionally characterized for bacterial blight disease resistance and nitrogen use efficiency (NUE) inrice.