| Bacillus cereus AR156 is a plant growth-promoting rhizobacterium originally isolated from the rhizosphere of tree soil from garden, Nanjing, China, can protect tomato against bacterial wilt caused by Ralstonia solanacearum and the root-knot nematode Meloidogyne incognita. Here, we investigate the mechanism of induces syatemic resistance by Bacillus cereus AR156 in greenhouses. We found that AR156 could elicit induced systemic resistance (ISR) against DC3000 on tomato. Furthermore, AR156 triggers ISR in Arabidopsis by simultaneously activating the SA- and JA/ET-signaling pathways in a NPR1-dependent manner, which leads to an additive effect on the level of induced protection. Meanwile, we discovered root exudation is an essential signal for induced systemic resistance by AR156. We found that miR863-3P could be highly induced by Pst DC3000 (avrRpt2) and miR863 play an important role in plant diesese resistance. To study the role of Small RNAs in induce systemic resistance by Bacillus cereus AR156, we collected the samples after bacterial pathogen Pseudomonas infection with and without Bacillus cereus AR156 pretreatment. Small RNA was extracted from Arabidopsis leaf samples and small RNA libraries were constructed. We will sequence these small RNA libraries using Illumina Hi-Seq deep sequencing and expect find some small RNAs are clearly differentially regulated by AR156 treatment. The projects that elucidate the molecular regulatory mechanism of induced systemic resistance and the knowledge gained from these studies will be instrumental in improving the application of AR156 to plant protection.1. Effect of biocontrol preparation AR156 combination against root-knot nematodes tomatoThe result showed that AR156 combination could promote the growth and yield of the plant after treated for 60 days. The growths of the plant and the number of fruiting have increased by 46.5% and 55.3%, respectively. Investigated at the end of the cropping season, the average biocontrol efficacy was 69.3% and yield increasement was 38.6% in the treatment of AR156 combination. Meanwhile, AR156 combination also could improve the quality of tomato, compared with the control. All the concentration of available N、P、K was all significantly after treated by AR156 combination.2. Induces systemic resistance by Bacillus cereus AR156 on tomatoWe investigate the ability of AR156 to promote plant growth and its role in the systemic protection of tomatoes cultivated in greenhouses against bacterial speck disease caused by Pseudomonas syringae pv. tomato DC3000 (DC3000). In our experiments, the AR156 population reached 105-106 CFU/g rhizosphere soil, and remained at that level in the roots of tomato plants for more than 2 months. In terms of its ability to promote plant growth, AR156 increased the average biomass of the tomato by 47.7%. AR156 also elicited induced systemic resistance (ISR) against DC3000, significantly reduced bacterial speck disease severity 1.6-fold, and inhibited proliferation of the pathogen by approximately 15-fold. This strain triggered the accumulation of defense-related genes in tomato leaves and primed the leaves for accelerated defense-related gene expression upon challenge with DC3000.3. The mechanism of inducing systemic resistance by Bacillus cereus AR156This study analyzed AR156-induced systemic resistance (ISR) to DC3000 in Arabidopsis ecotype Col-0 plants. Compared to mock-treated plants, AR156-treated ones showed an increase in biomass and reductions in disease severity and pathogen density in the leaves. The defense-related genes PR1, PR2, PR5 and PDF1.2 were concurrently expressed in the leaves of AR156-treated plants, suggesting simultaneous activation of the salicylic acid (SA)-and jasmonic acid (JA)/ethylene (ET)-dependent signaling pathways by AR156. The above gene expression was faster and stronger in plants treated with AR156 and inoculated with DC3000 than that in plants only inoculated with DC3000. Moreover, the cellular defense responses hydrogen peroxide accumulation and callose deposition were induced upon challenge inoculation in the leaves of Col-0 plants primed by AR156. Also, pretreatment with AR156 led to a higher level of induced protection against DC3000 in Col-0 than that in the transgenic NahG, the mutant jar1 or etr0; but the protection was absent in the mutant nprl. Therefore, AR156 triggers ISR in Arabidopsis by simultaneously activating the SA-and JA/ET-signaling pathways in a NPR1-dependent manner, which leads to an additive effect on the level of induced protection. To further study the mechanisms of AR156 induces systemic resistance, we speculated that AR156 and Arabidopsis roots interaction will produce some root exudates, which might play an important role in the induction of plant disease resistance. AR156 suspension was drenched into the soil around the Arabidopsis roots with or without activated charcoal, which can absorb root-exuded secondary compounds. Five days later, the plants were challenge inoculated with DC3000. AR156 pretreatment resulted in significant disease reduction. However, the induced resistance detected above was abolished after adding activated carbon. While the root colonization of AR156 was almost the same two lines. Thus, we deduced that the population of rhizobacteria AR156 did not influence the property of ISR when treatment with activated charcoal. These results suggest that the root exudation is an essential signal for induced systemic resistance by AR156.4. The role of small RNAs in disease resistanceWe found that miR863-3P could be highly induced by Pst DC3000 (avrRpt2) Compared to wild-type, the target At5g61570 overexpression plants were more susceptible to Pst DC3000 (avrRpt2). The double mutant was more disease-resistant to pathogen, but another target mutant se was more sensitive to Pst DC3000 (avrRpt2). So miR863 play an important role in plant diesese resistance. To study the role of Small RNAs in inducing systemic resistance by AR156, we harvested the leaves of Arabidopsis plants treated with AR156 alone and of those treated with AR156 and inoculated with DC3000, respectively. We expect to find the Small RNAs through high-throughput sequencing and bioinformatics analysis. Then we can study the mechanisms in disease resistance. Firstly, we extract the total RNA from the plant tissue and predicated the 20-30nt RNA. Second, we Link 3’ adapter ligation and 5’adapter ligation respectively. And Gel purification of ligated RNA product. We got cDNA library by RT-PCR. Lastly, we did TA colon and tested the quality of RNA libraries.5. ConclusionWe have gotten some new findings as followed:First, this is the first report one plant growth-promoting rhizobacteria (PGPR) via simultaneous activation of the SA-and JA/ET-signaling pathways resulting in an enhanced level of induced protection. Second, we found that miR863 play an important role in plant diesese resistance.However there are some shortages in this study:First, We did not study the defense signal on tomato due to the lack of mutant seeds. Second, we did not study deeply on the function of root exudates in induces systemic resistance by Bacillus cereus AR156. |
PDF Full Text Request