A novel pathogenicity gene is required in the rice blast fungus to suppress the basal defenses of the host

Abstract

For successful colonization and further reproduction in host plants, pathogens need to overcome the innate defenses of the plant. We demonstrate that a novel pathogenicity gene, DES1, in Magnaporthe oryzae regulates counter-defenses against host basal resistance. The DES1 gene was identified by screening for pathogenicity-defective mutants in a T-DNA insertional mutant library. Bioinformatic analysis revealed that this gene encodes a serine-rich protein that has unknown biochemical properties, and its homologs are strictly conserved in filamentous Ascomycetes. Targeted gene deletion of DES1 had no apparent effect on developmental morphogenesis, including vegetative growth, conidial germination, appressorium formation, and appressorium-mediated penetration. Conidial size of the mutant became smaller than that of the wild type, but the mutant displayed no defects on cell wall integrity. The Deltades1 mutant was hypersensitive to exogenous oxidative stress and the activity and transcription level of extracellular enzymes including peroxidases and laccases were severely decreased in the mutant. In addition, ferrous ion leakage was observed in the Deltades1 mutant. In the interaction with a susceptible rice cultivar, rice cells inoculated with the Deltades1 mutant exhibited strong defense responses accompanied by brown granules in primary infected cells, the accumulation of reactive oxygen species (ROS), the generation of autofluorescent materials, and PR gene induction in neighboring tissues. The Deltades1 mutant displayed a significant reduction in infectious hyphal extension, which caused a decrease in pathogenicity. Notably, the suppression of ROS generation by treatment with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases, resulted in a significant reduction in the defense responses in plant tissues challenged with the Deltades1 mutant. Furthermore, the Deltades1 mutant recovered its normal infectious growth in DPI-treated plant tissues. These results suggest that DES1 functions as a novel pathogenicity gene that regulates the activity of fungal proteins, compromising ROS-mediated plant defense.

Figure 1. The Magnaporthe oryzae T-DNA mutant ATMT0144A2 has defects in lesion development and conidial morphology.

(A) Rice seedlings (Nakdongbyeo) were inoculated with the wild-type strain 70-15 (left) and ATMT0144A2 (right). Diseased leaves were harvested 7 days after spray inoculation with conidial suspension (1×10⁵ conidia/ml). (B) Light microscopy of conidia produced by 70-15 (top) and ATMT0144A2 (bottom). Bar = 20 µm. (C) Conidial size of the wild type and ATMT0144A2. Values are the mean±SD from >100 conidia of each strain, which were measured using the Axiovision image analyzer. Length is the distance from the base to apex of conidia. Width is the size of the longest septum.

Figure 2. An abnormal T-DNA is integrated in the promoter region of MGG04163.

(A) Southern hybridization with ATMT0144A2. Total genomic DNA was digested with BglII and probed with the HpaI-digested HPH fragment. (B) Schematic diagram of T-DNA in ATMT0144A2. Specific primers used for the confirmation of T-DNA insertion (small arrows), vector read-through (slashed box) and unknown regions (dashed line) are indicated. The T-DNA insertion point is –750 from MGG04163 start codon. (C) Sequences of the T-DNA junction sites. Sequences of both junctions between T-DNA (upper case letters) and the M. oryzae genome (lower case letters) are indicated. Typical right-border cleavage site (white arrowhead), micro-homology region (bold), and a filler DNA (gray) are denoted. (D) Co-segregation of conidial morphology and T-DNA in F1 progeny. Seven-day-old conidia produced by randomly selected F1 progeny from ATMT0144A2×70-6 crosses were observed under a light microscope, and they were examined on TB3 medium containing 200 ppm hygromycin B. Bar = 10 µm. (E) The transcriptional expression of MGG04163 in ATMT0144A2. The transcription level of MGG04163 was assayed by quantitative RT-PCR using mycelia of the wild type and ATMT0144A2 in 3-day-old liquid culture.

Figure 3. The loss of DES1 leads to reduced pathogenicity and a colonization defect in host tissues.

(A) Pathogenicity assay. Five milliliters of conidial suspension (1×10⁵ conidia/ml) of each strain were sprayed on rice seedlings (Nakdongbyeo). Diseased leaves were harvested 7 days after inoculation. (B) The disease severity of each strain was assessed from the percentage diseased leaf area as calculated using the Axiovision image analyzer. Values are the mean±SD from eight rice leaves inoculated by each strain. (C) Observation of infectious growth. Excised rice sheath from 5-week-old rice seedlings (Nakdongbyeo) was inoculated with conidial suspension (1×10⁴ conidia/ml of each strain). Infectious growth was observed 96 h after inoculation. Bar = 50 µm.

Figure 4. The deletion of DES1 caused the induction of strong plant defense responses.

(A) DIC and fluorescence microscopy of infected rice sheaths (Nakdongbyeo) 48 h after inoculation. DIC images were captured using an 80-ms exposure time of transmission light with a DIC filter. Fluorescence images were captured using a 500-ms exposure for absorbed light using a GFP filter. Arrowheads on DAB staining panel indicate appressorium. Bar = 30 µm. (B) The expression of rice pathogenesis-related (PR) genes over time after inoculation. The transcriptional expression of PR1a and PBZ1 in the infected rice was analyzed using quantitative RT-PCR.

Figure 5. The inhibition of ROS generation recovers the infectious growth of the Δdes1 mutant.

(A) The excised sheath of rice (Nakdongbyeo) was inoculated with conidial suspension (1×10⁴ conidia/ml) of 70-15, Δdes1, or DES1^T-DNA with or without diphenyleneiodonium (DPI) dissolved in DMSO. Samples were harvested and observed 48 h after inoculation. Bar = 50 µm. (B) Percentage of appressorium-mediated penetration and infectious hyphae development of 70-15, Δdes1, and DES1^T-DNA in DPI treated onion epidermis. The total number of appressorium is indicated above each column. The level of IH development were scored after 72 h after inoculation (see Materials and Methods for details).

Figure 6. The Δdes1 mutant is hypersensitive to oxidative stress.

(A) Mycelial colonies on complete agar medium with or without 2–3 mM H₂O₂ on day 4 after inoculation. (B) Mycelial growth on complete medium with or without H₂O₂ (1–5 mM) on day 7 after inoculation. The colony diameters of four replicates were measured. Error bars represent the standard deviation.

Figure 7. DES1 is related to activity of extracellular peroxidase and laccase.

(A) The discoloration of Congo Red was tested on medium containing 100 ppm of the dye at final concentration. Strains were inoculated on CM agar medium containing Congo Red. Discoloration was observed on day 4. Left: wild type, middle: DES1^T-DNA, right: Δdes1. (B) Peroxidase activity measured by ABTS oxidizing test under H₂O₂ supplemented condition (see Materials and Methods for details). Black column: wild type, grey column: DES1^T-DNA, white column: Δdes1. (C) Laccase activity measured by ABTS oxidizing test without H₂O₂. The strain scheme is same with panel B. Error bars represent standard deviation.

Figure 8. Expression Profiles of M. oryzae Peroxidases in the Δdes1 Mutant.

A combination of the phylogenetic tree, expression characteristics, and domain architecture of 16 putative peroxidases in the M. oryzae genome were displayed. The phylogenetic tree was generated by ClustalW sequence alignment with 1000 bootstrappings and divided into three clades. The transcript levels of the the putative peroxidase encoding genes in the oxidative condition and/or in the Δdes1 mutant are indicated. Relative abundance of transcript compared with standard condition (wild type, normal condition) is displayed as a white triangle (up-regulated) or an inverted black triangle (down-regulated). Triangles indicating more than 2.0 (fold change) are displayed as trapezoids by cutting the top of the triangle. Fold changes of the standard condition (1.0) are not shown. Up-regulated genes in the Δdes1 mutant (more than 1.5 fold) were indicated in blue, and down-regulated genes in the Δdes1 mutant (less than 0.6 fold) were indicated in red. The InterPro terms and signal peptides are indicated (see legend).

Figure 9. DES1-eGFP is localized in vacuoles.

(A) Growing hyphae (left) and conidia (right) expressing DES1-eGFP in Czapek-Dox media. In the merged image, the original blue color from CMAC was changed to red for better visualization, so the co-localized spots were indicated as yellow. Bars = 10 µm. (B) Growing hyphae (left) and conidia (right) expressing eGFP without DES1 in Czapek-Dox media. Bars = 10 µm.