Infection Process Of Ustilaginoidea Virens And Host Rice Transcriptional Dynamic Change Revealed By DGE

Rice false smut is a typical fungal disease occurred in rice spikelets caused by Ustilaginoidea virens. In the last decades, with popularization of good rice cultivars and intensive application of nitrogen fertilizer, rice false smut is rapidly spreading around rice-growing regions worldwidely. In some areas, it has replaced rice blast and bacterial blight and becames a primary disease. Aparts causing severe yield losses, the toxins produced by false smut can greatly inhibit cell division which threatens to people life. However, the infection only occurs on booting spikelet strongly hinders the reaserch go on, thus the results mainly focon on the pathogen diversity, infection process, heredity, toxity and damage to rice production up to known. The infection process and molecular interaction between rice and pathogen are still poorly understood. In this study, we observed the infection process after inoculation and analyzed the expression profile of a spikelet infected with U. virens using digital gene expression tag profiling (DGE). Some interesting results were obtained as follow.1. Coleoptiles can be infected by U. virens. After inoculation rice bud with coindia, we obesved coleoptiles can be infected by pathogen at10day post inoculation (dpi). Several host cell were filled with swell spores and formed "spore stripes". Some spore germinated hyphe, grew alone with intercellular cell and entered host cell by directly pierced into cell wall. These results showed that false smut might be a systemic disease, and fungus carried by seed might be the source of primary infection.2. Cytological observation showed the early development of false smut took place in a stepwise invasion process and could be divided into three distinctive stages based on morphology of the infected floral organs and tissues. Initially, the two pistils in the infected spikelet were separate and only latter intertwined together by the hyphae, this phase here was named stage S1. As the fungi reproduced, mass of hyphae surrounded pistils and formed a penetration peg on column. Then, visible mycelial mass reached at the bottom of anthers, hyphae climbed up the anther using some special organs like "feet" and "feelers", this stage was here named stage S2. As the disease continued to develop, the whole floral organ became surrounded by mycelium, forming a floral-hyphae complex. Finally, the hyphae entered the anther chambers by piercing through anther wall at the weak connectivum, this stage was here named stage S3. Cytological observation showed mass of conidia wrapped on the surfaces of pollen grains and innate anther wall, and some of pollen became collapsed. Carbohydrate analysis showed the content of soluble sugar increased significantly whereas starch content significantly reduced. This was further confirmed by the decrease of expression level of starch synthase and increase of the starch-degrading enzyme glycosyl hydrolase. In addition, two sugar transporter members were also highly induced in the present study, hinting stamen is an important tissue for U. virens infection and reproduction.3. By integrative two years (2010-2011) DGE data, totally423,879and1620DE genes across both years were monitored in S1, S2and S3, respectively. To obtain the genes specially regulated by false smut, we compared our data to that of the openly available biotic and abiotic databases to exclude genes generally regulated by other abiotic and biotic factors by bioinformatics.126(58up,68down),304(136up,168down), and580(243up,373down) expressional genes which be considered specially regulated by the invasion of U. virens were detected in S1, S2, and S3respectively. Of which,53and68core genes were detected among three infection stages in up-and down-regulation part.4. Analysis of the highly conserved c/s-acting elements between core up-and down-regulation genes, we obtained9elements (6up and3down). In up parts, YTCANTYY and TTATTT are basic transcription initiation elements for genes expression, TTGAC, TGACT for W-box, TGTCA for Myb and TAAAG for Dof were typically stress related motifs. In down parts, two (CANNTG and CATGCA) out of three motifs were detected for abscisic acid (ABA)-response. Previous studies showed ABA is usually a positive regulator in response to abiotic stress, whereas a negative one in response to biotic stress. CGCG box is regulated by calmodulin. As an important second message, calmodulin play a key role in plant development.5. GO analysis showed mass of caterious differently enriched. Of which,4categories in "Cell part",55categories in "Molecular functions",52categories in "Biological process". Further analysis showed in the beginning infection,"Phosphorylation" and "Protein modification" were greatly activated whereas "Carbohydrate metabolic process" was repressed. With disease development, categories as "Cell death" was widely up-regulated whereas "Ion transport" ere widely repressed at stage S2. During final infection (S3),"Cell recognition" was enriched in up-regulation and "Cell wall metabolism" was enriched in down-regulation part, which was in accordance with the cytological observation that the pathogen hyphae intertwined with microspores and great amount of conidia produced in the anther chambers.