Transcriptome analysis of Lr19-virulent mutants provides clues for the AvrLr19 of Puccinia triticina

Abstract

Introduction: Wheat leaf rust caused by Puccinia triticina (Pt) remains one of the most destructive diseases of common wheat worldwide. Understanding the pathogenicity mechanisms of Pt is important to control wheat leaf rust.

Methods: The urediniospores of Pt race PHNT (wheat leaf rust resistance gene Lr19-avirulent isolate) were mutagenized with ethyl methanesulfonate (EMS), and two Lr19-virulent mutants named M1 and M2 were isolated. RNA sequencing was performed on samples collected from wheat cultivars Chinese Spring and TcLr19 infected with wild-type (WT) PHNT, M1, and M2 isolates at 14 days post-inoculation (dpi), respectively. Screening AvrLr19 candidates by quantitative reverse transcription PCR (qPCR) and Agrobacterium-mediated transient assays in Nicotiana benthamiana.

Results: 560 genes with single nucleotide polymorphisms (SNPs) and insertions or deletions (Indels) from non-differentially expressed genes were identified. Among them, 10 secreted proteins were screened based on their fragments per kilobase of exon model per million mapped reads (FPKM) values in the database. qPCR results showed that the expression profiles of 7 secreted proteins including PTTG_27471, PTTG_12441, PTTG_28324, PTTG_26499, PTTG_06910, PTTG_26516, and PTTG_03570 among 10 secreted proteins in mutants were significantly different with that in wild-type isolate after infection wheat TcLr19 and might be related to the recognition between Lr19 and AvrLr19. In addition, a total of 216 differentially expressed genes (DEGs) were obtained from three different sample comparisons including M1-vs-WT, M2-vs-WT, and M1-vs-M2. Among 216 DEGs, 15 were predicted to be secreted proteins. One secreted protein named PTTG_04779 could inhibit programmed progress of cell death (PCD) induced by apoptosis-controlling genes B-cell lymphoma-2 associated X protein (BAX) on Nicotiana benthamiana, indicating that it might play a virulence function in plant. Taken together, total 8 secreted proteins, PTTG_04779, PTTG_27471, PTTG_12441, PTTG_28324, PTTG_26499, PTTG_06910, PTTG_26516, PTTG_03570 are identified as AvrLr19 candidates.

Discussion: Our results showed that a large number of genes participate in the interaction between Pt and TcLr19, which will provide valuable resources for the identification of AvrLr19 candidates and pathogenesis-related genes.

Keywords: AvrLr19 candidates; Puccinia triticina; sequence insertion or deletion; single nucleotide polymorphism; wheat.

Figure 1

Lr19 provides prehaustoria resistance against Pt. (A) Infected leaves of susceptible cultivar Chinese Spring (Lr19-) and resistant line TcLr19 (Lr19+) were observed; NC: negative control, non-inoculation. (B) Fungal infection hyphae (IH) (stained blue) entered the leaf mesophyll tissue (stained blue) through the plant stoma (S) in both wheat lines at 24 hpi. Fungal haustoria (H) developed only in susceptible Chinese Spring. Imaging at 48 hpi revealed fungal growth in susceptible Chinese Spring, but no further fungal growth in TcLr19. At 48 hpi revealed presumably dead host cells (stained green) in TcLr19; no dead cells were revealed in Chinese Spring. U, spores; AP, appressorium. Scale bars, 100 μm.

Figure 2

Lr19-virulent Mutants were obtained and confirmed. (A) The germination effect of Pt spores of PHNT race under different EMS concentration; CK, control check. (B) The phenotypes of Lr19-virulent Mutants M1 and M2 were compared with wild-type (WT). (C) Molecular markers detection of Lr19. There was a specific band at 500 bp for forward primer detection and there was no specific band at 750 bp for reverse primer detection, M is the abbreviation of marker, 1–9 represents 9 test samples. (D) Heatmap of WT and its mutants (M1 and M2) of Pt based on infection types, the virulence characterization of all isolates was conducted on the 36 wheat Lr single-gene differentials. Its 0 to 4 were transformed to the color key ranging from green to red, which indicate avirulent (resistant) to virulent (susceptible) reactions.

Figure 3

The types and numbers of SNPs in M1, M2, and WT among not differentially expressed genes. (A) Numbers of SNPs from the M1-VS-WT and M2-VS-WT. (B) Numbers of Indels from the M1-VS-WT and M2-VS-WT. (C) Results of KEGG analysis of secreted proteins. (D) GO annotation for secreted proteins.

Figure 4

The genes containing SNPs and Indels with different expression patterns were selected. (A) Their expression patterns were generated based on corresponding FPKM values from the transcriptome database. The transcript levels of the genes containing SNPs and Indels selected during Pt infection at 4, 6, and 8 days post-inoculation (dpi). (B–D) Transcriptional profile of genes during the Pt pathotypes infection measured by qPCR. The transcript levels of the 14 secreted proteins during Pt infection at 0, 12, 48, and 96 hpi (Hour post inoculation) were determined by qPCR assay. The transcript levels for all genes were expressed as linearized fold-Pt actin levels, which were calculated according to the formula 2^−△CT. Data were expressed as mean values ± SE from four biological replicates. An asterisk (*p < 0.05,**p < 0.01) indicates.

Figure 5

The differentially expressed genes (DEGs). (A) Number of up-regulated and down regulated DEGs indifferent samples. (B) DEGs that are unique or shared among various samples comparisons in mutants (M1, M2) and WT. The numbers of DEGs are noted in each section of the Venn diagrams. (C) Bar graph showing the number of 216 DEGs identified utilizing gene ontology (GO) enrichment analysis for involvement in specific molecular function, cellular component and biological processes. In the figure, red color represents molecular function, green color represents molecular function, blue color represents molecular function, the Y coordinate is the name of GO term, and the X coordinate is number of gene.

Figure 6

Characterization of secreted protein PTTG_04779. (A) PTTG_04779 can suppress BAX-induced cell death. PTTG_04779 was infiltrated into N. benthamiana leaves, followed 24 h later by infiltration with A. tumefaciens carrying the Bax gene. pCamA and buffer served as a negative. (B) Transcriptional profile of genes during the Pt pathotypes infection measured by qPCR. (C) Expressed fluorescently tagged PTTG_04779_ΔSP location in the N. benthamiana leaves epidermal cells. The pCamA-PTTG_04779_ΔSP (with GFP tag) in N. benthamiana cells and GFP field show green. Scale bar, 50 μm. (D) Yeast secretion trap assay of the secreted protein PTTG_04779. The predicted signal peptide coding sequences of PTTG_04779 (PTTG_04779_SP) were cloned into the yeast secretion trap vector pSUC2. The Ps87 was used as a positive control, empty vector and Mg87 was used as a negative control.