Characterization of E3 ubiquitin ligase genes in rice
By Blastp searches against the rice genome database using the characterized 11 Arabidopsis genes as queries, corresponding genes were obtained. Phylogenetic tree analysis revealed that Os04g34030, Os02g33590 and Os01g64570 respectively showed similarity to Arabidopsis AtPUB22, AtPUB23 and AtPUB24 which were already reported to have functions in response to biotic and abiotic stresses. Os06g13870 showed similarity to AtPUB21, TdPUB21 and HvPUB21. Os06g34390 showed similarity to SbATL41 and ZmATL6, and Os05g01940 and AtATL9 were gathered in one cluster (Figure 1).
The expression levels of Os06g13870, Os04g34030 and Os02g33590 were strongly induced by the inoculation of M. oryzae and hormone molecules
To test whether these 11 genes have functions on responses to stress, the expression patterns of these genes with inoculation of M. grisea and treatment of hormone molecules were analyzed. As showed in Figure 2a, the expression of Os06g13870, Os04g34030 and Os02g33590 was strongly induced by the inoculation of M. grisea in incompatible interaction, while other genes not. The expression levels of these genes were analyzed with the treatment of hormone. As showed in Figure 2b, the expression levels of Os06g13870, Os04g34030 and Os02g33590 were strongly induced by SA, JA and ACC while the expression levels of other genes not. ABA is a well-known stress-related hormones in plants and involved in the responses to biotic and abiotic stress. We test the expression patterns of E3 ubiquitin ligase genes with ABA treatment. In the treatment of ABA, the expression levels showed no significant difference from control except Os06g34390 and Os05g01940 (Figure 2c).
The expression patterns of E3 ubiquitin ligase genes in response to abiotic stress
As reported, PUB genes were involved in the response to abiotic stresses containing drought, cold, heat and salt to different degrees (Lu et al. 2020). So drought, salt, cold and heat stresses were selected for the analysis of the expression patterns in abiotic stress. In the drought stress, almost all the genes had no changes except two genes, Os06g34390 and Os02g33590. They increased dramatically 2 hours after treatment (Figure 3a). In the cold stress, the expression levels of all the genes showed no significant difference from control except Os05g01940 which was strongly induced 12 hours after treatment (Figure 3b). In the heat and salt stresses, the expression levels of all genes showed no significant from control after treatment (Figure 3c-d).
BMV:Os06g13870-infiltrated plants showed decreased resistance to M. grisea when compared with BMV:00-infiltrated plants, while BMV:Os04g34030- and BMV:Os02g33590-infiltrated plants showed increased resistance
We explored the possible function of these genes in the resistance to M. grisea by comparing the phenotype of BMV:target genes- and BMV:00-infiltrated plants after the inoculation of M. grisea. The silencing efficiency was tested before inoculation and the really silencing plants were selected for disease assay (Figure 4a). After 7 days, the BMV:Os06g13870-infiltrated plants showed severer disease phenotype with larger lesion size and more fungi growth when compared with control while BMV:Os04g34030- and BMV:Os02g33590-infiltrated plants showed lighter disease phenotype with smaller lesion size and less fungi growth (Figure 4b-d).
In order to explore the mechanism of Os06g13870, Os04g34030 and Os02g33590’s function in the resistance to M. grisea, we analyzed the condition of ROS accumulation and the expression levels of defense-related genes. First, we analyzed the condition of ROS accumulation. As showed in Figure 5a, there was no significant difference among BMV:Os06g13870-, BMV:Os04g34030-, BMV:Os02g33590- and BMV:00-infiltrated seedlings before M. grisea inoculation. While after M. grisea inoculation, the BMV:Os04g34030- and BMV:02g33590-infiltrated seedlings accumulated less ROS than BMV:00-infiltrated seedlings, while BMV:Os06g13870- infiltrated seedlings accumulated more. H2O2 content showed similar results. After M. grisea inoculation, H2O2 content in BMV:Os04g34030- and BMV:02g33590- infiltrated seedlings is lower than that in BMV:00-infiltrated seedlings while higher in BMV:06g13870-infiltrated seedlings (Figure 5b). SOD activity and CAT activity were analyzed to explore the reason for the changed H2O2 content in BMV:Os06g13870-, BMV:Os04g34030- and BMV:Os02g33590-infiltrated seedlings. As showed in Figure 5c-d, before M. grisea inoculation, SOD activity and CAT activity in BMV:target genes- and BMV:00-infiltrated seedlings showed no significant difference. After M. grisea inoculation, SOD activity in BMV:Os04g34030- and BMV:02g33590- infiltrated seedlings decreased while CAT activity increased when compared with BMV:00-infiltrated seedlings (Figure 5c-d). SOD activity in BMV:Os06g13870- infiltrated seedlings increased while CAT activity decreased when compared with BMV:00-infiltrated seedlings after M. grisea inoculation (Figure 5c-d).
Second, we analyzed the expression levels of defense-related genes. As showed in Figure 6, the expression levels of OsLOX1, OsPR3, OsNH1, OsPR1a and OsWRKY45 decreased in BMV:Os06g13870-infiltrated plants when compared to control after M. grisea inoculation while increased in BMV:Os04g34030- and BMV:Os02g33590- infiltrated seedlings. These results indicated Os06g13870, Os04g34030 and Os02g33590 involved in the resistance to M. grisea, may through regulating the accumulation of ROS and the expression of defense-related genes.
The BMV:Os02g33590-infiltrated plants increased the tolerance to drought stress while BMV:Os06g34390-infiltrated plants decreased the tolerance to drought stress
To explore the possible function of these 11 genes in response to abiotic stress, we compared the phenotype of BMV:target gene- and BMV:00-infiltrated plants after suffered from abiotic stress. None had dramatic difference from control expect BMV:Os06g34390- and BMV:Os02g33590-infiltrated plants. The BMV:Os02g33590- infiltrated plants showed increased tolerance while BMV:Os06g34390-infiltrated plants showed decreased tolerance to drought when compared with control (Figure 7a and 8a). Water loss and survival rate further confirmed this conclusion. Water loss in BMV:Os06g34390-infiltrated plants is higher than control while lower in BMV:Os02g33590-infiltrated plants (Figure 7b, 8b). The survival rate, proline content, and sugar content decreased dramatically in BMV:Os06g34390-infiltrated plants (Figure 7c-e) while increased dramatically in BMV:Os02g33590-infiltrated plants (Figure 8c-e). We also tested the expression levels of drought-responsive genes. The expression levels of drought-responsive genes decreased in BMV:Os06g34390- infiltrated plants while increased dramatically in BMV:Os02g33590-infiltrated plants (Figure 9).
BMV:Os05g01940-infiltrated plants decreased the tolerance to cold stress
In the cold stress, BMV:Os05g01940-infiltrated plants showed decreased resistance when compared with control (Figure 10a). The survival rate of BMV:Os05g01940- infiltrated plants was 19.62% , while 83.65% in control (Figure 10b). The MDA content and electrolyte leakage in BMV:Os05g01940-infiltrated plants increased when compared with control (Figure 10c-d) while chlorophyll content in BMV:Os05g01940-infiltrated plants decreased (Figure 10e). The expression levels of cold-responsive genes were analyzed next. As showed in Figure 10f, the expression levels of cold-responsive genes decreased significantly in BMV:Os05g01940- infiltrated plants when compared with control.