| Bananas and plantains are one of the important crops in tropical and subtropical areas, which have been regarded as the forth largest food crops after rice, wheat and corn. However, during the growth, banana usually suffered from various stresses, such as pathogen, typhoon, low temperature and drought. Recently, Panama disease threatened the banana production which was caused by soil-borne fungi, Fusarium oxysporum f. sp. cubense (Foe), especially, the race 4 of Foe threatened most varieties of banana. What's more serious is that there is no efficient fungicide or soil fumigant to control this disease. The farmer only used the inefficient method to prevent and control this disease, which can not satisfy the development of banana industry. Therefore, we must know the mechanism of banana susceptible to Foe TR4. In this study, we used banana varieties (Musa acuminata L. AAA group, cv. Brazilian) susceptible to study the mechanism of banana-Fdc TR4 interaction.In the study of the mechanism of banana susceptible to Foe TR4, we mainly researched the progress of the pathogen infection of the banana roots, the manner of infection, physiological and biochemical changes of banana roots. Acquire the whole transcriptom of the banana by using RNA-seq, and then, the differential expression genes generated by digital gene expression profile after inoculation Foe TR4 in banana roots. Primarily know about the reasons of banana susceptible to Foe TR4. According to the results of the analysis, we further validate the mechanism of salicylic acid inducing the banana resistance to Foe TR4 infection. The main results were shown as follows:(1) Using GFP-tagged fungi, it could be viewed in vivo and the dynamic pathogen process. In 2 DAI (2 day after inoculation), the fungi adhered to the banana roots in 3 forms:hypha, large conidia and small conidia. In 4 and 6 DAI, Foe TR4 attacked the epidermal cells and into the inner of roots. Most hypha grew between gaps of cells. And the cell death appeared before the hypha attached in the banana roots. At the same time, the physiological and biochemical enzymes was induced at the 2 DAI and 4 DAI, such as CAT, APX, SOD, POD, PAL, MDA and H2O2. These factors can be used as a good biochemical detection of banana-Foe TR4 interaction.(2) Acquire and analyze the banana roots transcriptom. Sequencing the library at 0 day,2 day,4 day and 6 day, totally acquire 26,662,006 reads and 2,399,580,540 bp nucleotides. The mean length of all reads was 90 bp. After filtrating the low quantity and shorter than 500 bp generated in assembling, acquire 25,158 Unigenes, the number larger than 2000 bp was 5166, the mean length was 1439 bp.17,540 Unigenes were annotated by using GO (Gene Ontology), and 11611 Unigenes were annotated by using KEGG (Kyoto Encyclopedia of Genes and Genomes). These results indicate that the study acquired a high quality banana root transcriptom.(3) Analyze the differential expression genes in banana roots after inoculation with Foc TR4. The four libraries were sequenced and generated above 14,381,501 primary Tags. Delete the low quality sequence Tags,3,254,559 to 3,561,806 Tags were acquired in each library. Map all these Tags into the banana root transcroptom before acquirement. Comparing the inoculation samples and the non-inoculation sample, set FDR< 0.001 and log2Ratio? 1 as a threshold value to determine the genes differentially expressed in two periods. There were 4,729,5,078 and 5,531 genes differentially expressed respectively in 2 DAI,4 DAI and 6 DAI. All these genes were analyzed by using GO enrichment, the condition of enrichment was FDR adjusted P value ? 0.05. Interestingly, all the down expression genes were not enriched in any GO terms. There were enriched 8,22,11 GO terms in 2,4,6 DAI respectively. All differentially expressed genes mapped to the KEGG pathway. Down expression of genes were not enriched in any pathways in KEGG There were 5,3,5 metabolism pathways enriched in 2,4,6 DAI respectively. All these results indicated that salicylic acid biosynthesis and signal-transduction pathways were suppressed, which was one of the reasons for banana susceptible to Foc TR4.(4) Salicylic acid was involved in banana resistance to Foc TR4 and the mechanism of exogenous salicylic acid induced resistance of the banana. Under the whole banana roots transcriptom,31 sequences were acquired, which were the key enzyme genes associated in shikimate pathway and Phenylpropanoid biosynthetic pathway. Shikimate pathway and Phenylpropanoid of biosynthetic pathway were activated in resistant variety after inoculation Foc TR4, while those genes were suppressed in susceptible variety. The salicylic acid signal transduction related genes were activated in resistant variety and inactivated in susceptible variety, so the salicylic acid metabolism was correlated with the resistance of banana. Using 100O?M concentration SA to pretreat 60 day old bananas seedlings for 2 days, and then inoculated Foc TR4, observed the disease development. There was no appearance of disease symptom in SA pretreatment. The growth of seedlings were as the same as mock treatment. In internal symptoms, salicylic acid treatment of banana seedlings rhizome did not find significant browning. SA treatment can induce banana disease resistance. Exogenous salicylic acid can change within salicylic acid biosynthetic after inoculation Foc TR4. Exogenous Salicylic Acid fully activate the shikimate pathway and phenylpropanoid biosynthesis pathway related genes and salicylic acid biosynthesis and signal transduction related genes, suggesting that exogenous salicylic acid could change endogenous salicylic acid to improve the banana resistance.In summary, this study, based on physiology, starts from transcriptom to analyze the mechanism of banana susceptible to Foc TR4. And further identify the reason of banana susceptible to Foc TR4 was that the salicylic acid biosynthesis and signal transduction pathway were suppressed. The exogenous salicylic acid could enhance the banana resistance to Foc TR4. These results not only enrich the molecular mechanism of the banana-Foc TR4 interaction, but also provide a new method for the prevention and treatment of fusarium wilt of banana. |
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