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, 7 (4), e1001335

HYR1-mediated Detoxification of Reactive Oxygen Species Is Required for Full Virulence in the Rice Blast Fungus


HYR1-mediated Detoxification of Reactive Oxygen Species Is Required for Full Virulence in the Rice Blast Fungus

Kun Huang et al. PLoS Pathog.


During plant-pathogen interactions, the plant may mount several types of defense responses to either block the pathogen completely or ameliorate the amount of disease. Such responses include release of reactive oxygen species (ROS) to attack the pathogen, as well as formation of cell wall appositions (CWAs) to physically block pathogen penetration. A successful pathogen will likely have its own ROS detoxification mechanisms to cope with this inhospitable environment. Here, we report one such candidate mechanism in the rice blast fungus, Magnaporthe oryzae, governed by a gene we refer to as MoHYR1. This gene (MGG_07460) encodes a glutathione peroxidase (GSHPx) domain, and its homologue in yeast was reported to specifically detoxify phospholipid peroxides. To characterize this gene in M. oryzae, we generated a deletion mutantΔhyr1 which showed growth inhibition with increased amounts of hydrogen peroxide (H₂O₂). Moreover, we observed that the fungal mutants had a decreased ability to tolerate ROS generated by a susceptible plant, including ROS found associated with CWAs. Ultimately, this resulted in significantly smaller lesion sizes on both barley and rice. In order to determine how this gene interacts with other (ROS) scavenging-related genes in M. oryzae, we compared expression levels of ten genes in mutant versus wild type with and without H₂O₂. Our results indicated that the HYR1 gene was important for allowing the fungus to tolerate H₂O₂ in vitro and in planta and that this ability was directly related to fungal virulence.

Conflict of interest statement

The authors have declared that no competing interests exist.


Figure 1
Figure 1. MoHYR1 is a putative thioredoxin peroxidase protein and highly conserved among filamentous fungi.
(A) A Prosite search of the amino acid sequence revealed two glutathione peroxidase domains, the first of which is an active site, and the second, a signature (image was drawn with DomainDraw, [45]). (B) Alignment of the M. oryzae HYR1 with nine filamentous fungi. Shaded boxes below the alignment indicate degree of conservation. Open boxes indicate locations of domains in A. Arrows indicate the conserved cysteines. (C) Dendrogram of HYR1 from eleven filamentous fungi, one copy from yeast and one from human.
Figure 2
Figure 2. M. oryzae HYR1 shares similar tertiary structure with yeast HYR1.
The predicted tertiary structure of MoHYR1 from M. oryzae was constructed with the PyMOL program. Helices, sheets and termini are tentatively labeled according to the yeast HYR1 structure; the two connecting cysteines are in red, while the cysteine (Cys 39) that would form an intermolecular bond with the HYR1-interaction protein, YAP1, is shown in purple and labeled.
Figure 3
Figure 3. MoHYR1 complements the S. cerevisiae Δhyr1 mutant.
The yeast strains BY4741 (wild type) and BY4741 YIR037W (Δhyr1) were obtained from the ATCC. The mutant was complemented with the wild type copy of itself, the MoHYR1 gene, and the MoHYR1 containing mutations at each of the two cysteine residues (cys39Ala and cys88Ala). All strains were spotted onto YPD plates containing 0 mM, 2 mM and 4 mM hydrogen peroxide. Neither the YIR037W strain, nor the two cysteine residue mutants grow at the non-permissive concentration however the yeast mutant is partially rescued by the MoHYR1 copy. This experiment was repeated ten times with similar results.
Figure 4
Figure 4. Δhyr1 exhibits a virulence defect.
Δhyr1 mutants display a decrease in pathogenicity compared to wild type, on susceptible barley and rice. (A) Conidia of two Δhyr1 mutants, B25 and B33, were drop-inoculated onto barley cultivar Lacey and show a virulence defect compared to ectopics (B40 and B60), the complemented line (Δhyr1 - C), or 70-15 (wild type), as manifested by smaller lesions at 7dpi. (B) Quantification of lesion size reveals a significant difference in virulence between wild type and ectopics, and the mutants. Different letters over the bars indicate a significant difference as determined by a student's t-test, and a p-value of ≤0.01. (C) Rice plants (cultivar Maratelli) were spray-inoculated with the mutants, ectopics and wild type (as above) and scored for lesion type 7 dpi. (D) Quantification of lesion type (0 = no symptom; 1 = pinhead-sized brown specks; 2 = 1.5 mm brown spots; 3 = 2–3 mm gray spots with brown margins; 4 =  many elliptical gray spots longer than 3 mm; 5 = coalesced lesions infecting 50% or more of the leaf area), reveals no difference in lesion types 1-3 however the two mutants do not make any lesion types 4 and 5. Lesions were photographed and measured or scored 7dpi and experiments were repeated twice with similar results. Different letters over the bars indicate a significant difference as determined by a student's t-test and a p-value of <0.05.
Figure 5
Figure 5. More ROS was produced when leaves were inoculated with Δhyr1 mutant conidia, versus wild type.
(A) Conidia of wild type (70-15), (B) ectopic (B40) and (C) Δhyr1 (B25) were inoculated onto the surface of a barley leaf and then stained with calcofluor white for fungal cell walls and DCF for the ROS, 24 hpi and imaged by confocal microscopy. (D) Around 35 Appressoria were counted for each line, along with the number of appressoria showing ROS haloes, and percentages were generated. This experiment was repeated ten times with similar results. Different letters over the bars indicate a significant difference as determined by a student's t-test, and a p-value of ≤0.05. Scale bar  = 20 µm for all images.
Figure 6
Figure 6. The ROS observed after inoculation with Δhyr1 conidia as a disk-shaped halo located beneath appressoria.
(A) A 3-D projection of confocal images with the ROS stain H2DCFDA showed a halo (green) of ROS around and beneath the appressoria (blue; AP), which emanated from two nearby conidia. (B) A side-view of panel A showed that the halo was a thin layer of ROS located beneath the appressoria. The ROS halo sits directly between the AP and the plant surface. Scale bar  = 10 µm.
Figure 7
Figure 7. Δhyr1 (B25) conidia on gel-bond were similar to wild type in terms of ROS production.
Staining was performed 24 hpi; Calcofluor White was used to stain the cell walls (blue) and H2DCFDA was used to stain the ROS (green). Conidia of (A) Δhyr1 (B25), (B) wild type (70-15) and (C) ectopic (B40). A transmitted light image was taken as well, and overlaid with the fluorescent image. The inset in panel A showed the fluorescence image of the conidium (1) and appressorium (2). Images were taken using confocal microscopy. Scale bar  = 10 µm.
Figure 8
Figure 8. Δhyr1 appressorial-localized ROS appeared to be plant-generated.
(A) Reflection confocal imaging with the ROS stain DAB shows a wide ROS signal (arrow) around and beneath the appressorial attachment site (AP). In the middle of the appressorium attachment site was the putative penetration peg site (arrowhead). (B) The same interaction site as Fig. 8A, embedded in epoxy resin and imaged under confocal microscopy revealed DAB deposited (arrow) beneath and surrounding an attempted penetration site (arrowhead). The deposit was located up against the plant cell wall (PC) on the inside of the cell. Scale bar  = 5 µm.
Figure 9
Figure 9. ROS scavenging in the plant rescued the hyr1 mutant phenotype.
(A) Conidia of Δhyr1 (B25) were mixed with 0.5 mM ascorbic acid and inoculated onto the leaf surface. Infected leaves were stained for ROS 24 hpi. (B) Conidia were mixed with water and inoculated onto the leaf surface. Leaves were first treated with 0.5 mM ascorbic acid for 1 hour and stained for ROS 24 hpi. (C) From left to right: Δhyr1 (B25), Δhyr1 (B33) (where susceptible barley leaves were treated with 0.5 mM ascorbic acid for 1 hour and then inoculated with mutant spores in water) ectopic (B40), ectopic (B60), wild type (70-15). Scale bar  = 20 µm for all confocal images.
Figure 10
Figure 10. Mutants have more DAB staining than wild type revealed a stronger plant reaction.
DAB staining was performed on wild type (70-15) conidia (A, C, E) and Δhyr1 (B25) mutant conidia (B, D, F) 24hpi. Wild type (70-15) conidia on the leaf surface shows DAB staining mostly the fungal structures while Δhyr1 (B25) mutant conidia elicit a stronger ROS plant reaction. Images were generated with a transmitted light microscope. Scale bars  = 100 µm.
Figure 11
Figure 11. Two plant defense responses overlap when the Δhyr1 mutant conidia were inoculated onto leaves.
Correlative images show plant reaction underneath appressoria 24 hpi. (A) ROS staining; (B) aniline blue staining; (C) merged image of panels A and B. Images were processed sequentially (ROS followed by aniline blue), imaged by confocal microscopy and correlated. Scale bar  = 2.5 µm.
Figure 12
Figure 12. Putative plant-generated cell wall appositions surround the penetration sites 40 hpi.
Confocal 3-D maximum intensity projections of aniline blue stained infected leaves showed cell wall appositions. (A) A representative cell wall apposition (yellow) shown here was detected in barley 40 hpi with Δhyr1 (B25) mutant conidia. (B) Comparable cell wall appositions (yellow) were also detected in barley 40 hpi after inoculation with wild type (70-15). Transmitted light images were merged with 3-D confocal data to aid in visualization of plant and fungal structures. Scale bars  = 5 µm.
Figure 13
Figure 13. Antioxidant gene orthologs have altered expression in the Δhyr1 mutant versus wild type.
Wild type (70-15) and Δhyr1 mutant (B25) were grown in 0 mM and 5 mM hydrogen peroxide and collected 1 hour after immersion. RNA was extracted and real-time qRT-PCR performed on three biological replicates. (A) The YAP1, GTO1, GLR1 and GSH1 all increase in expression in wild type upon H2O2 challenge, but the latter three display low levels in the mutant. (B) CAT1, SOD1, GSH2, GTT1 and cyt c peroxidase do not display significant changes in expression. MoHYR1 expression is abolished in the mutants. Letters over bars represent statistically significant differences between expression changes of the genes (statistics were generated using student t-test with p-value <0.05).
Figure 14
Figure 14. MoHYR1 changed localization during pre-penetration events on the surface of a leaf.
The MoHYR1 coding sequence was fused to the cerulean fluorescent protein to study protein localization during early infection. (A) HYR1 at 1 hpi with putative vacuole location and low level cytoplasmic distribution; the germ tubes has formed, but no appressorium. (B) HYR1 at 6 hpi with increased cytoplasmic localization where it is likely to be required to function in ROS scavenging; an immature appressorium was apparent. (C) HYR1 at 12 hpi with cytoplasmic location; a mature appressorium was apparent. (D) HYR1 at 24 hpi with vacuole and low level cytoplasmic localization in the appressorium. (E) HYR1 at 72 hpi again showing cytoplasmic localization. Images were taken with confocal microscopy and all experiments were done on the surface of barley leaves. Scale bar shown  = 10 µm for all images.

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