1 INTRODUCTION
Wheat is critical to food security worldwide. Tilletia laevisKühn lead to common bunt, which seriously reduce wheat grain quality and yield in wheat growing regions of the world (Albughobeish et al., 2015), the average yield losses by the bunt pathogens can reach 70-80% in some wheat producing areas (Goates et al., 2012). Additionally, wheat dwarf bunt caused by Tilletia controversa Kühn , the teliospores or bunt sori of the T. controversa can last for 10 years in soil and are easily dispersed from field to field with soil, or can blowout to new places by infected wheat seeds (Gao et al., 2014). The bunt sori have a strong odor like of rotting fish, which heavily degrade the quality of the wheat seeds and flour (Gaudet et al., 2007). Even slightly low infection can result in clear smell in flour minced from infected grains (Chen et al., 2016; Nguyen et al., 2019). Also, T. controversa is an internationally important quarantine pathogen and many countries have strict restriction on importing wheat from areas where the disease occurs (Mathre et al., 1996; Gao et al., 2014). Both pathogens grow vigorously and disrupt the normal structure of wheat anther (JIA et al., 2013; Bokore et al., 2019), which can infect reproductive organs at different stages of anther development causing some of the most economically important plant diseases (Ngugi & Scherm, 2006).
In every anther lobe, cell differentiation and division produce pollen mother cells (PMC), which are bounded by four somatic layers, epidermis (EPI), endothecium (EN), middle layer (ML) and tapetum (TA) from the outside to inside (Scott et al., 2004; Chang et al., 2011; Xu et al., 2016; Browne et al., 2018). EPI prevent water loss and have role in dehiscence and in gas exchange, EN and ML have a role in cell structure and support to the other cells (Goldberg et al., 1993; Sun et al., 2007). TA plays vital roles in supporting pollen grain development succeeding the completion of meiosis and meiotic cytokinesis (Zhang et al., 2006; Browne et al., 2018). Additionally, TA is the innermost layer of the anthers and have a role to provides the nutrients to the emerging pollen grain and it undergoes to program cell death in the later stages of pollen grain development. Microspores (MP) are involved in the pollen grain and sperm cell development (Zhang & Wilsonc, 2009). Anther is particularly more sensitive to biotic and abiotic factors (Hedhlyet al., 2009) during development (Saini & Westgate, 1999; Dolferus et al., 2011). Ustilago maydis causes disturbance in the anther lobes and infected organs (Walbot & Skibbe, 2010) which changed the cells of anther lobes of maize i.e. EPI, EN, SPL, ML and TA, which are involved in the development of pollen mother cells (Gao et al., 2013).RAFTIN have a role in TA development of cereal crops (Wang et al., 2003). MYB transcription factors have a role in anther development in barley and other cereal crops (Murray et al., 2003).TaMYB80 influence the pollen grain development in participating the program cell death of TA cells (Gao et al., 2013; Millar & Gubler, 2005; Xue, 2005)
High-throughput gene expression analysis using RNA-Seq signifies a very powerful tool for transcriptomic description of plants during the interaction of pathogens and plants (Wang et al., 2009; Massart et al., 2014). The RNA-Seq has been performed in many crops against different pathogens, such as banana and Fusarium oxysporum f. sp.cubense (Li, 2013), mango and Fusarium mangiferae (Liu et al., 2016), potato and Phytophthora infestants (Gao et al., 2013) and so on.
Virus-induced gene silencing (VIGS) has been developed as an effective genetics tool for assessing gene functions in different plants species including maize, pea, wheat, tobacco, tomato and barley (Constantin et al., 2004; Scofield et al., 2005; Ding et al., 2006). Previous studies revealed functional analysis of wheat genes against Zymoseptoria tritici resistance and susceptibility (Lee et al., 2015) used forLr21 -mediated leaf rust resistance pathway in hexaploid wheat (Gao et al., 2013), and established in wheat spikes and grains for HMW-GS-encoding genes, in which clear photobleaching symptoms were appeared in silenced plants (Ma et al., 2012).
In this study, we used confocal microscopy to build up the timeline of wheat anther development, which showed the characteristics of cell types in wheat anther, and the anther timeline was approved by section and quantification of anther developmental genes using qRT-PCR assays. In addition, RNA-Seq was used to analyze wheat anther stages transcriptome changes in response to T. laevis and T. controversainfection. VIGS was used to silence one of anther development genes in RNA-Seq to explore the function of anther genes in the infection process of T. laevis and T. controversa . This is the first report of the bunt pathogens reprogrammed the anther development based on confocal microscope and RNA-Seq analysis, and explore related gene’s function by VIGS technique.