THTT and THTS expressed complicated expression patterns when interacting with Thatcher at 144hpi
Pathotypes expressed in many differences in the interaction process with Thatcher when we detected at 144 hpi, including 1,076 genes that were significantly up-regulated of the 10,483 up-regulated genes and 1,708 genes that were significantly down-regulated of the 10,689 down-regulated genes in (THTT) compared to (THTS). We aligned 566 significantly up-regulated genes and 1,225 down-regulated genes to KEGG and GO. Of these 2,784 genes with significantly different expression, 100 genes were present in the Nr NCBI Library, and 78 genes of them were significantly up-regulated, while 22 genes were significantly down-regulated in THTT compared to THTS. A GO analysis was performed to classify these differentially expressed genes into 3 major biological categories. These genes belonged to 19 classes involved in biological processes, 9 classes involved in cell location categories and 12 classes involved in molecular functions.
According to the RNA-seq library, CL622.Contig3 was annotated as mannosyl-oligosaccharide glucosidase (OsMOGS). OsMOGS was involved in N-glycan synthesis in rice, which played an important role in establishing and maintaining the growth of auxin and the growth and development of the root system . Therefore, we speculated that this gene participated in the development of epithelial cells and mycelium growth.
Unigene1676 is annotated to ubiquitin E3 ligase. E3 ubiquitin ligases transfer Ub to one or more Lys residues in the substrate by linking the C-terminal Gly of Ub with a Lys of the target protein (and/or a Lys of the Ub itself). Ubiquitin E3 ligase is closely related to the regulation of the cell cycle, tumourigenesis, cell proliferation, cell apoptosis, signal transduction, cell growth, cell immunity, inflammation and the regulation of the replication and repair of DNA. F-box protein (SCF) E3 ligases are the largest E3 gene family, of which the F-box protein is the key component to determine substrate specificity. Some studies have revealed that the deletion mutant of GrrA, a F-box protein in Aspergillus nidulans is unable to produce mature ascospores because of a block in meiosis .
GTPases (singular GTPase) are a large family of hydrolaseenzymes that can bind and hydrolyze GTP. The GTP binding and hydrolysis takes place in the highly conserved G domain common to all GTPases. Zhang et al.  verified the functions of all six Rho GTPases in Fusarium graminearum by constructed the deletion vectors. Mutation △Fgrac1, △Fgcdc42 and △Fgrho4 were drastically reduced in growth rate and △Fgrho4 lacked aerial hyphae. △Fgrho2 and △Fgrho3 were less drastic on CM plates compared to other mutants. FgRho1 is essential for fungal survival. FgRho2, FgRho4, FgCdc42 and FgRac1 were involved in sexual development and pathogenesis while FgRho2 and FgRho4 were both involved in cell wall integrity, only FgRho4 showed a role in nuclear division and septum formation. The knocking down of TaRab7 enhanced the susceptibility of wheat Suwon 11 to an avirulent race CYR23, which implies that TaRab7 plays an important role in the early stage of wheat-stripe rust fungus interaction and in stress tolerance . In our research the Unigene17170_Tc15_2 peaked at 12 hpi in THTT and peaked at 48-72 hpi in THTS when forming appressorium, it may be involved in the formation of hyphae.
qRT-PCR revealed more detail differential expressed patterns at early stage between THTT and THTS
The results of the qRT-PCR were almost same as the patterns in the RNA-seq at 144 hpi. However, there are more differential expressed patterns at early stage. Twelve tested genes have obvious peak expression before 144 hpi (Fig 3).
Expression profiles of 9 genes found in THTT precedes that in THTS. CL3900.Contig1 peaked at 6 hpi both in THTT and THTS. CL3499.Contig1 peaked at 6 hpi in THTT and 36 hpi in THTS. CL6956.Contig1 peaked at 6 hpi in THTT, which is much higher than that of THTS.
Unigene18070 peaked at 12 hpi in THTT and 24 hpi in THTS. CL2376.Contig1 peaked at 12 hpi in THTT and 24 hpi and 48 hpi in THTS. The quantity of Unigene17170 increased and peaked at 12 hpi, but it peaked at 48 hpi in THTS with higher quantity than that in THTT. Unigene1676 peaked at 12 hpi with the expression quantity three times of that in THTS. Unigene11683 has two peaks at 12 hpi and 36 hpi in THTT and one peak at 48 hpi in THTS. The higher expression of Unigene18727 is at 12 hpi in THTT.
Expression profiles of 4 genes found in THTS precedes that in THTT. CL622.Contig3 expressed at 6 hpi, and the quantity of CL622.Contig3 was higher than that in THTS. Unigene22186 had two peaks-12 hpi and 144 hpi in THTS while one peak at 144 hpi in THTT. Unigene11935 peaked at 12 hpi and 36 hpi in THTS with more than 100 times of the quantity in THTT. Unigene14763 had almost same expression pattern in two races.
Unigene18727 was found with conserved domain of cytochrome P450 52A6. CYP53 family members play a key role in fungal colonization of plant material by detoxification of anti-fungal compounds released by plants or generated during plant material degradation. Moreover, CYP53 family members play a role in the generation of a secondary metabolite, veratryl alcohol, which is crucial in the degradation of the plant cell wall component, lignin . The expression of Unigene18727_Tc15_2 peaked in THTT at 12 hpi and gradually increases and peaks at 72 hpi in THTS, Further, it hardly expresses at later stage. Combining with the function of cytochrome P450, we assume that the gene helps infect host. And this gene expressed earlier and stranger than that in THTS.
CL2376.Contig1 is a member of ATP binding cassette transporter C family. The ABC transporter belongs to a large ancient protein family Energy produced by ATP binding and hydrolysis is used for the ABC transporter participation in the substrate transport process, such as the RNA translation and transmembrane process that is required for DNA repair. Although the mechanism is unclear, it plays an important role in enhancing the ability of the pathogen to resist adverse external environment. CL2376.Contig1_Tc15_2 had two peaks, 24 hpi and 48 hpi in THTS, and had only one peak at 12hpi in THTT, and the expressed quantity was significant higher than that in THTS, so it possibly plays part in pathogenic process. Chitinase can degrade most fungal cell walls, prevent or interrupt fungal infection, colonization and expansion in plants. Chitin deacetylase (CAD) belongs to chitinase. The enhanced CGA activity in the process of the formation of appressorium in Uromyces and appressorium formation failure in the polycarbonate (PC) artificial culture medium caused by CAD deletion mutants in Magnaporthe oryzae both indicate that CAD is involved in the formation of appressorium . Furthermore, CAD has a promoting effect on the formation of fungal fruiting body. CAD isolated from the basidiomycetes of Flammulina velutipes specifically express during the fruiting stage .
CL3499.Contig2_Tc15_2 was predicated to encode chitin deacetylase. This gene expressed up regulatedly and peaked at 6 hpi in THTT and peaked at 72 hpi in THTS, expressed more earlier than that in THTS. Therefore, we speculated that the CL3499.Contig2_Tc15_2 gene may play a key role in the pathogenic infection of leaf rusts.
Effector Unigene22186 is with superoxide dismutase (SOD) activity. Jeong et al.  found that MnSOD role in organisms facing exogenous oxidative ROS and especially superoxide overproduction. The role of MnSOD was deduced from the SOD2 increased expression after exposure to menadione in S. pombe. Furthermore, SOD2 mutants were more sensitive than wild type to menadione, and plumbagin, a menadione derivative. SOD2 plays an important role in the virulence of both C. neoformans var. gattii and C. neoformans var. grubii, depending on the route of infection [33,34]. Since defense against ROS is determinant for pathogenicity, MnSOD evolutionary history and pathophysiological roles in invasive or allergic mycoses agree with the hypothesis that pathogenicity emerged multiple times within fungi emerged multiple times within fungi. MnSOD was used for taxonomic and evolutionary data . This gene expressed in THTT and up regulated from 6hpi and peaked at 144 hpi, however it was expressed in THTS had two peaks at 12hpi and 144 hpi respectively and the expressed quantity was higher than that in THTT. We guess that Unigene22186 is a disease-related gene.
There are many factors that lead to the difference of the two strains. In order to explore the reasons for the toxicity difference of these two strains, we will focus on these specific expression genes in future experiments.
Effectors secreted by THTT and THTS when interacted with Thatcher have different expression patterns
Functional domains were obtained by Interpro Scan to make clear the biological functions of the 54 candidate secreted proteins (Table S6), including rare lipoprotein A (RlpA)-like domain superfamily, osmotin or thaumatin-like superfamily, glycoside hydrolase family 7, concanavalin A-like lectin, Kre9/Knh1 family, UDP-glucose: glycoprotein glucosyltransferase.
Candidate effector Unigene15605 was up regulated in THTS. It was found with Kre9/Knh1 family related to cell wall organisation in fungi. Gilbert et al.  have identified that the H99 deletion mutants kre5Δ and kre6Δskn1Δ contained lessβ‐1,6‐glucan, which is the component of cell wall, grew slowly with an aberrant morphology. Moreover, these two mutants resulted in alterations in cell wall chitosan and the exopolysaccharide capsule, a primary cryptococcal virulence determinant.
25-66AA of Unigene11683 is similar with concanavalin A-like lectin family. Sequence analysis were conducted to show that the N-terminal regions of BEACH proteins shares similarities with concanavalin A (ConA)-like lectin superfamily while members of the BEACH family are generally defined as trafficking regulatory of vesicle, a transport carrier in the process of secretory protein . However, 21-69AA of the gene is aligned with glycoside hydrolase family 7. Van et al.  constructed knock-down (KD) mutants of cellulases in order to clarify that cellulases, which can hydrolyze crystalline cellulose to permeate the host epidermis belong to glycosyl hydrolase (GH) families 6 and 7. The results showed that transcript levels of the target genes and cellulase activity were considerably reduced in the KD mutants, and the KD mutants resulted in fewer lesions, less penetration, and infection of fewer cells compared with the parent strain.
Fifty-four candidates differentially expressed effectors and expression profiles of 4 candidate effectors were obtained and two candidate effectors were identified in this study. These results lay the foundation for the study of the pathogenic differences in Pt at the molecular level and reveal the interaction mechanisms. Furthermore, this study is mainly based on bioinformatics, macroscopic screening and qRT-PCR technology, and the specific differentially expressed genes should be further investigated. Other members of our experimental group have performed studies using gene silencing techniques and tobacco inoculation of the pathogenic genes to verify their function.