Differential Expression Profiling Of Two Different Pathotypes Of Puccinia Triticinia With Different Virulence And Screening Of Secreted Proteins

Wheat leaf rust fungi（Puccinia triticinia） is one of the devastating crop pathogens. To develop resistant plants, a better understanding of rust virulence factors, or effector proteins, is needed. Races of P. triticina perform differences in pathogenicity when interact with wheat,because of the pathogenic specificity of different Pt races. To ascertain the molecular mechanism of pathogenicity is of great significance to revealing the different virulence of wheat leaf rust disease and for using resistance genes. In this study, two Pt pathotypes with different virulence, coded 08-5-361-1（THTT） and09-12-284-1（THTS）, were inoculated into susceptible wheat cultivar Thacher（Tc）. Total RNA was extracted from infected leaves when the inoculation part filled with haustoria.Two RNA samples, named Tc284₂ and Tc361₁, were sequenced by BGI Shenzhen company with digital expression profile sequencing on Illumina Solexa. To erase the genes came from host Tc, we filtered the results of samples Tc361-1 and Tc284-2 reads by using database Tc15-2 and wheat（Chinese spring） as reference genome. Secretory proteins and candidate effectors were found by bioinformatics methods analysis. These results established the foundation for elucidating the cause of different expression of genes between virulence races and effectively undertake the progress of previous research in our laboratory.The main results are as follows:1. The digital express gene library of Pt races 08-5-361-1 and 09-12-284-1 were established. The two races were inoculated to Tc respectively, and the RNA isolated 6 days post inoculation was sequenced by The BGI Company. RNA from both the host and fungi were present and separated by alignment to the Pt RNA transcriptomes library of Tc15₂and the transcriptomes of Tc284₂ and Tc361₁ were established. In sample Tc284₂,353,131,779 total base pairs, 7,206,771 total reads, 2,935,991 total mapped reads（40.74%）,2,375,423 unique matched（32.96%） were gained by comparing to reference Tc15₂ database. In sample Tc361₁, 344,976,954 total base pairs, 7,040,346 total reads,2,476,977 total mapped reads（35.18%）, 1,994,137 unique match（28.32%） were gained by comparing to reference Tc15₂ database. We gained 19,950 unigenes from assembly data of Tc284₂, in which 10,593 unigenes were annotated to KEGG database. 19,138 unigenes were annotated to Blast NR. 3,296 unigenes were annotated to GO Process, 6,685 unigenes were annotated to GO Function, 5,633 unigenes were annotated to GO component items. And we gained 19,806 unigenes from assembly of Tc361₁, in which 10,657 unigenes were annotated to KEGG database. 18,973 unigenes were annotated to Blast nr. 5,720 unigenes were annotated to GO process, 6,739 unigenes were annotated to GO function, 3,379 unigenes were annotated to GO component items.2. Analysis the differential expressed genes between Tc284-2 and Tc361₁. tookTc284-2 as control group, and Tc361-1 as treatment group, a total of 2,784 differentially expressed genes（DEGs）（ the FDR（false discovery rate） ≤ 0.001 and less than 2 times of the difference of multiple genes） were gained, of which 1,708 were up-regulated, and1,076 down regulated. The up-regulated genes were annotated to MAPK signaling pathway, regulation of transcription, sequence-specific DNA binding transcription factor activity, small GTPase mediated signal transduction, RNA transport, substrate-specific transmembrane transporter activity, Lysine degradation, nucleic acid binding, zinc ion binding, asparagine synthase, glutamine-hydrolyzing activity, coenzyme binding, GTPase activity, ATP-dependent helicase activity, response to stress. The down-regulated genes were annotated to putative ABC transport system ATP-binding protein, intramolecular transferase activity, H+-transporting and cation-transporting ATPase activity, cell wall integrity and stress response component, misfolded or incompletely synthesized protein catabolic process, cytochrome-b5 reductase, Lon-like ATP-dependent protease, serine-type endopeptidase activity, organic cyclic compound binding, metal ion binding.3. Candidate secreted protein in the differential expressed genes were screened.Bioinformatics methods were used to analyze the proteins coded by the differently expressed genes inorder to screen the secreted proteins（SP）secreted by Pt. The result showed that 199 proteins contained the signal peptide by signal P 4.1 solftware analysis,4 proteins located in the mitochondria by target P 1.1 server solftware analysis, 40 proteins contained the transmembrane domain by TMHMM Server v. 2.0. 5 proteins with GPI anchor modification sites were forecast by Big-PI, excluding the proteins of mitochondria-target, contained the transmembrane domain, GPI anchor modification sites. 150 candidate secreted proteins from differently expressed genes were screened.Compared to Tc284-2, there were 92 up-regulated genes, and 58 down-regulated genes.The secreted proteins only expressed in Tc284-2 were Unigene11683_Tc15₂,Unigene11935_Tc15₂, in Tc361-1 were CL4323 Contig1_Tc15₂, CL5606Contig1_Tc15₂, Unigene26432_Tc15₂ respectively.4. The structure of the 150 candidate secreted protein were predicted. Based on the secreted proteins we further used Pfam to predict the frameworks and functions of protein domains, and 36 proteins were predicted with Pfam notes. The remaining114 secreted proteins were screened and predicted de novo structure by using MEME Version 4.10.0 software, the results showed that 3 conserved domains(Yxxxx NKx DC,（Y[N/A] Y [T/D] [H/A] [C/A] [S/H] [T/Q] [S/N] [S/G] [N/C]T [N/A]W, [S/K] M [H/A] R[R/M] R [P/A] [W/Q]） were found in 15 secreted proteins, and the remaining 99 secretory proteins may contain new motifs. We gained 51 candidate effectors finally in differentially expressed genes.