Fig 1.
Growth promotion and root colonization in wild-type rice and symbiotic mutants by A. brasilense.
(A, D, G) show that total plant mass (mg) increased in wild-type rice, pollux, and dmi3 mutants upon inoculation with A. brasilense. Data represents the average of five experimental replications (n = 10–15) +/- SE. Asterisk (*) denotes significance between the conditions by t-test (P < 0.001, P < 0.001, P < 0.06). (B, E, H) show that root mass (mg) increased in wild-type rice, pollux, and dmi3 mutants upon inoculation with A. brasilense. Data represents the average of five experimental replications (n = 10–15) +/- SE. Asterisk (*) denotes significance between the conditions by t-test (P < 0.003, P < 0.01, P < 0.007). (C, F, I) show comparison of total colony-forming units (cfu) of A. brasilense determined by serial dilution and plate counts of bacteria between non-surface sterilized and surface sterilized roots of wild-type, pollux, and dmi3 rice seedlings inoculated with A. brasilense. The data are average of five experiments. Each experiment had at least three plants. Asterisk (*) denotes significance between the conditions by t-test (P < 0.001, P < 0.001, P < 0.001).
Fig 2.
Summary plots for the gene expression profiles and differential expression analysis.
Panels A and B show scatter plots of the first two principal components of the FPKM normalized gene expression profiles at 1dpi and 14dpi, respectively. Panels C and D respectively show volcano plots at 1dpi and 14dpi where mean log2 fold change is plotted against the –log10 FDR adjusted P-values for all expressed genes. Significant DE genes (FDR<0.05 and |FC|>2) were highlighted with red color.
Fig 3.
Bar plots summarizing the gene ontology terms over-represented in DE genes between control and A. brasilense samples for (A) 1dpi (B) 14dpi (C) common in both 1dpi and 14dpi datasets.
The magnitude of bars in the positive and negative directions respectively represents the proportion of up-regulated and down-regulated genes associated with each GO term. The width of each bar is proportional to the number of DE genes associated with the GO term. Blue dots represent –log10(FDR) for each significant GO term. A vertical dotted line at –log10(0.05) = 1.3 indicates the significance threshold. Bar color indicates the three categories of GO term: Biological Processes (BP), Molecular Functions (MF), and Cellular Components (CC).
Fig 4.
RT-PCR validation of differentially expressed genes identified by RNA-Seq.
Expression pattern of six DEGs was validated by RT-PCR. For the RT-PCR experiments, C1 and T1, C14 and T14 represent cDNA templates synthesized from control and treatment RNA samples at 1- and 14dpi respectively. RT-PCR was performed in at least three biological replicates for all the samples. Cyclophilin was used as an internal reference gene.
Fig 5.
Overview of the putative molecular signaling pathway in plant roots during interactions with Azospirillum brasilense.
The figure outlines a putative molecular signaling pathway in rice roots during interactions with A. brasilense. LYKs = LysM-RLK proteins with an active kinase domain, AP2/ERFs = APETALA 2/Ethylene response factors, NAC = NAM, ATAF1/2, and CUC, GRAS = GAI, RGA, and SCR, PR = pathogenesis-related.