Histology Of Oidium Heveae-Rubber Tree Interaction And Preli-Minary Analysis Of The Pathogen Genome

Powdery mildew in rubber tree, caused by obligate parasitic fungus Oidium heveae B A. Steinmann, significantly impacts rubber production. Yet, the study of of powdery mildew in rubber tree and genomics and transcriptomics involved in this process, very little research have been done on O. heveae. This study used powdery mildew strain HO-73derived in our lab by single spot separation and purification as well as Brazilian rubber tree clone7-33-97as a study model for host-pathogen interactions. We conducted cytological of rubber tree-powdery mildew interactions during infection and do the analysis based on the genomic sequencing.The main results as follows:1. Observed the morphology of conidia and examined the cytological changes in the germination process of O. heveae, further reveal they characters. The size of rubber tree powdery mildew conidia is26.1～45.1μm×13.5～21.9μm; they have oval or nearly spherical shape. Germination is not influenced by the contact surface, since conidia can form germ tubes and appressoria both in deionized water and in glucose solution. Energy for germination comes from conidia’s own reserves of glycogen and lipids, which can be transferred to appressorium via germ tube. Similarly, nucleolus can be transferred to appressorium. During conidium germination, several vacuoles can fuse into one and later they can dissipate so that no obvious vacuole structure can be observed.2. Systematic observed cytological changes and the physiological responses of host cells to the O. heveae in early infection stages, and reveals the pathogenic process. O. heveae development in the host goes through5crucial time points:conidium germination peak at4hours post infection (hpi); appressorium formation peak (8hpi); infection structure (primary haustorium) formation peak (15hpi); secondary hyphae formation peak (24hpi); conidiophores formation peak at5days post infection. In the interaction process, rubber tree cells respond to pathogen invasion only after the formation of primary haustoria. These resistance reactions include oxygen burst as well as callus and papillae formation. Reactive oxygen species play an important role in rubber tree-pathogen interactions. Low levels of reactive oxygen species allow O. heveae development and entry, while high levels of reactive oxygen species lead to oxygen burst which prevents further pathogen expansion. However, we did not detect a link between oxygen burst and expression of defense-related genes in the host.3. Explored out the high efficient RNA extraction and isolation methods to HO-73strain for different purpose. The powdery mildew conidia collected by the writing brush were used for RNA isolation. Three different methods were used for effective extraction of good-quality RNA, which had OD2.0-2.2and clear presence of28S and18S bands. When using a small amount of powdery mildew conidia, a modified Triozol RNA extraction method was clearly superior to other two methods. However, for powdery mildew samples obtained with5%acetyl cellulose dissolved in acetone, good quality RNA can be obtained with a RNA extraction kit (Omega, USA). Regarding two other methods, due to the presence of phenol in lysates, after liquid nitrogen treatment and grinding, precipitates are formed, making RNA extraction impossible.4. Finally, we conducted genomic and transcriptomic prelimary analyses of O.heveae. Predicted size of powdery mildew genome is60M; assembled genome has a size of58.5M (97.5%). Theoretically, a detailed map of powdery mildew genome has been already obtained; it has33192open reading frames. In terms of Biological Process genes (level3); only60are classified as pathogenesis genes, which is less than1.5%of overall genes. GC content is38.1%. GO annotation identifies4457genes;275genes are involved in the KEGG metabolic pathway. Pathway network map and preliminary location analysis were done based on KEGG database. Rubber tree powdery mildew, as a parasitic fungus that depends on its host in vivo, also exhibits the phenomenon of second level metabolic pathway gene deletion, such as the lack of genes for enzymes involved in cellulose, xylan and pectin degradation. Tryptophan dimethylallyl transferase (DMATS), polyketide synthases (PKSs), nonribosomal peptide synthetases (NRPSs) are also missing or are reduced in the overall genome.