4.4 RNA-Seq data in response to T. laevis and T. controversa
Pathogenesis-related (PR) genes and WRKY transcription factors play important role in defense mechanism and in regulation of defense genes (Anguelova‐Merhar et al., 2001; Sels et al., 2008; Pandey & Somssich, 2009; Rushton et al., 2010; Bai et al., 2018). We compared the transcription level between both pathogens infected and control plants at different wheat anther stages. Our results showed that transcriptional levels of 81 PR genes after T. laevis and 52 PR genes after T. controversa were changed. UDT1 is a transcription factor that plays a key role in the development of the tapetum and is required for anther male fertility and pollen differentiation (v et al., 2005; Liu et al., 2010). Additionally, the transcription factor TDR is involved in the regulation of programmed cell death (PCD) of tapetum and degradation of male reproductive development (Li et al., 2006). TDR plays an upstream role in the regulation of tapetum PCD and interacts with EAT1/DTD (Niu et al., 2013) and may also play a regulatory role in lipid metabolism involved in pollen wall formation (Zhang et al., 2008). In this study, the transcription factors UDT1, TDR and EAT1 were expressed at all stages of anthers development and TDR mostly down-regulated at fifth stage (1100-1300 µm) support hypothesis that tapetum cells degraded in the last stages. Polygalacturonase is a cell wall hydrolytic enzyme, which is related to various cell separation processes during plant development including; seed germination, anther dehiscence, organ shedding and fruit ripening (Kim & Patterson, 2006; Ogawa et al., 2009). In our study, the expression of polygalacturonase gene was down regulated at all stages of anther development (700-1100 µm), indicating that both pathogens alters the normal structure of anther development as a result anthers lose their pollination activities.
According to GO enrichment analysis of differentially expressed genes in control and both pathogens infected libraries, the results showed that binding, catalytic activity, membrane part, cell part, metabolic process and cellular process were had the highest number of DEGs during plant pathogen interaction (Figure S4 ). GO enrichment analysis suggest that both pathogens alters the morpho-physiological and defense mechanisms of the plants (Berger et al., 2007). KEGG is database resource for understanding high level functions and utilities of a biological system. This is especially useful for large scale molecular datasets generated by genome sequencing and other high-throughput experiment technologies, providing valuable classification for studies of biological complex process (Kanehisa & Goto, 2000). KEGG enrichment analysis showed that metabolic and cellular pathways were expressed significantly at all above anther stages after T. laevis andT. controversa infection. The photosynthesis is a basic physiological processes of plant growth (Deng et al., 2009). The results obtained from KEGG analysis of DEGs showed that genes involved photosynthesis significantly changed after T. laevis and T. controversa infection (Figure S5 ). Phenylalanine metabolism are the substrate molecules of primary and secondary metabolic pathways and have role in the viability of all cells (Adams et al., 2019). Endoplasmic reticulum are important organelle and have a key role for protein production destined for the plasma membrane, vacuoles and apoplast in plants (Eichmann & Schäfer, 2012). Plants hormones are the key regulators for many functions including primary (plant growth and development) and secondary (induction of defence programme) (Bolwell, 1997; Yvon & Joanne, 2011). Starch and sucrose have a central role in the carbon budget of the majority of plants, and plays a dual role in carbon allocation (MacNeill et al., 2017). Glycolysis have a major role in respiration rate of the plants by producing the substrates that are essential for respiration (Megguer et al., 2017). Our results showed that KEGG analysis of DEGs showed that the genes involved in phenylalanine metabolism, endoplasmic reticulum, plant hormone signal transduction, starch, sucrose and glycolysis were significantly changed after T. laevis and T. controversa infection in different stages of anther development (Figure S5 ).