Identification of M. oryzae Endo-1,4-beta-xylanase I and generation of ∆Moxyl1 strains
Domain-specific BLASTp search for the Neurospora crassa glycoside hydrolase family 11 domain amino acid sequence identified two GH11 family domain-containing proteins in Magnaporthe oryzae – MoXYL1A, encoded by MGG_07955, and MoXYL1B encoded by MGG_08424. To elucidate the physiological and pathological functions of MoXYL1A and MoXYL1B in M. oryzae, we generated targeted gene knock-out strains by replacing the coding region of MoXYL1A and MoXYL1B with the hygromycin phosphotransferase resistance (hph) gene using established homologous recombination techniques (Catlett, Lee, Yoder, & Turgeon, 2003). Putative MoXYL1A and MoXYL1B gene deletion transformants were selected on double layered TB3 agar containing 300 µg/mL (bottom layer) and 600 µg/mL (upper layer) hygromycin B and screened by PCR. Two successful knock-out strains each for MoXYL1A (∆Moxyl1A-3 and ∆Moxyl1A-13), and MoXYL1B (∆Moxyl1B-5 and ∆Moxyl1B-7) identified by PCR screening were checked using qRT-PCR and Southern Blotting (Supplementary Figure 1). These assays confirmed the successful replacement of the MoXYL1A and MoXYL1B genes with hph in these strains (Supplementary Figure 1). Our ability to recover deletion mutants indicates that survival of the rice blast fungus is independent of MoXYL1A and MoXYL1B function under standard conditions.
Influence of MoXYL1A and MoXYL1B gene deletion on vegetative and asexual growth of M. oryzae
To investigate the role of MoXYL1 gene deletion on the growth of M. oryzae, mycelial plugs of single and double ΔMoxyl1A and ΔMoxyl1B mutants, wild type (Guy11) and the complemented mutant strains were inoculated on Complete Medium (CM) and incubated under dark conditions at 28℃ for 10 days. Growth measurements (mm) were taken on day 10 post-inoculation and plates were photographed. This assay showed no strong adverse effects on growth for all strains tested (Figure 1a-b, Supplementary Figure 2a-b). However, a noticeable reduction in aerial hyphae and minimal but statistically significant difference in colony diameter was observed for ΔMoxyl1A compared to WT. In contrast, there was no significant difference between ΔMoxyl1B and WT. The double deletion strain (DKO) was obtained via HR-based deletion of MoXYL1B on the ΔMoxyl1A background. Colony morphology and size of the double mutant was not significantly different from either single knockout. We conclude that MoXYL1A and MoXYL1B do not have specific morphogenesis-related functions in blast fungus under standard conditions.
A conidiophorogenesis assay was conducted to ascertain the impact of these mutants on asexual reproduction in M. oryzae, as conidiation plays a vital role in the survival and dissemination of the fungus (He, Wang, Chu, Feng, & Ying, 2015). Small plugs of the mutant and wild type strains, along with the complemented and double knock out strains were grown on rice bran medium plates and incubated under dark conditions at 28℃ for seven days. On day 7, the hyphae were scratched and incubated under light conditions, and conidiophore production was visually observed at 12h, 24h, 36h, and 48h. To quantify conidia production, conidia were harvested after 10 days, diluted with an optimized volume of sterile distilled water and then counted using a hemocytometer. The results showed that the ΔMoxyl1A and ΔMoxyl1A/ΔMoxyl1B strains were severely impaired in conidiophore production, with almost no conidia produced, while ΔMoxyl1B produced conidiophores of WT shape but in reduced number relative to WT (Figure 1d). To further corroborate this defect, mutant and wild type strains were also grown on SDC and CM-II media and conidia were counted (Supplementary Figure 2). The results confirmed a significant reduction in spore production in the deletion mutants, with a complete lack of conidiation in the double mutant, suggesting that there is clear contribution of these genes to the asexual development of M. oryzae, with MoXYL1A having an essential role and MoXYL1B a partial role in this growth phase. The conidiation defect of ΔMoxyl1A was partially rescued in the complemented strain; however, although it produced conidia that are morphologically indistinguishable from the wild type, the overall number was reduced. The defective conidiation was fully restored in the MoXYL1B-complemented strain (Supplementary Figure 2).
MoXYL1A is required for complete virulence of M. oryzae
A susceptible rice cultivar CO39 and detached leaves of the Golden Promise cultivar of barley were used to conduct pathogenicity assays to assess the role of MoXYL1 genes in the pathogenesis of rice blast fungus. Fungal mycelia cultured in liquid CM media from wild type, mutants, complementation, and double deletion strains were inoculated on intact and abraded detached leaves of barley and kept under proper humidity conditions at 28℃ for 24 h of darkness preceded by six days exposure to light. This assay showed that fungal virulence was impaired in ΔMoxyl1A and DKO strains, which were unable to produce proficient blast lesions, while ΔMoxyl1B produced typical blast lesions (Figure 2a). This suggests that MoXYL1B is dispensable for pathogenicity while MoXYL1A plays a significant role in imparting virulence to M. oryzae. A comparable experiment was done using a spore suspension inoculated onto intact and abraded barley leaves. We infected with conidia of WT, single mutants, and complemented strains and observed similar pathogenicity defects for ΔMoxyl1A conidia as were observed with mycelia. Virulence defects were rescued in the complemented strain (Figure 2a). These results support the key role of MoXYL1A in the pathogenicity of rice blast disease on barley.
We further conducted inoculation trials with spore suspensions (1 × 105 conidia per ml in an aqueous solution of 0.2% Tween 20) on rice (cultivar CO39). Spore suspensions of wild type Guy11 and MoXYL1A or MoXYL1B mutant strains were independently and evenly sprayed on rice leaves, and plants were kept under proper incubation conditions (see Methods) for seven days. This rice pathogenicity trial showed consistent results with the barley experiments, with ΔMoxyl1A and ΔMoxyl1A/ΔMoxyl1B strains completely lacking virulence as compared to wild type and complemented strains (Figure 2b).
To unravel the factors responsible for the impairment in pathogenicity of the MoXYL1A deletion mutants, we performed a penetration bioassay using barley as the host plant. We inoculated barley leaves obtained from one-week-old barley plants with conidia harvested from ΔMoxyl1A and wild type Guy11 to examine the penetration ability and colonization efficiency of the fungus. The results showed that the targeted gene replacement of MoXYL1A had a profound impact on the penetration and likely colonization abilities of M. oryzae as compared to wild type. At 48hpi, for ΔMoxyl1A, no invasive hyphae were visualized inside the barley leaf when its sheath was excised and observed under the microscope, while WT micrographs showed pronounced invasive hyphae that were branched and colonizing adjacent cells. These results confirmed the inability of MoXYL1A mutants to invade host plants and cause blast disease. Consistent with earlier results, no penetration defects were observed for the MoXYL1B deletion (Figure 2c).
To further investigate the reason for pathogenicity defect observed in the ΔMoxyl1A strain, we performed an appressorium formation assay to assess the efficiency of pathogenic dirrentiation in the ΔMoxyl1A, and ΔMoxyl1B strains compared to the wild-type and the complementation strain. The ΔMoxyl1A strain was unable to form a normal appressorium at 8h of incubation on hydrophobic coverslips. The mutant produced an abnormal appressorium with a long germ tube and no melanin-ring, suggesting that it was a non-functional appressorium that could not penetrate and colonize the barley leaves (Figure 2d). This phenotype was rescued by complementation of MoXYL1A. The MoXYL1B deletion mutant strains also had delayed appressorium formation but their appressoria were morphologically normal. As the double deletion mutants are unable to form conidia, we could not assess their appressorium development.
ΔMoxyl1A and ΔMoxyl1B are sensitive to cell wall stress
Fungal cell wall integrity is crucial for infection of host cells, as the fungal cell wall maintains shape and facilitates exchange between the environment and fungus (Cabib, Roh, Schmidt, Crotti, & Varma, 2001). For proper growth and development, the cell wall requires repeated remodeling (Jeon et al., 2008). Therefore, we set out to assess the impact of cell wall-perturbing reagents on the growth of ΔMoxyl1 strains. Calcofluor White (CFW) is used to test whether fungal strains are defective in cell wall assembly or have a defect in cell wall integrity (Lussier et al., 1997; Ram, Wolters, Hoopen, & Klis, 1994). Sodium dodecyl sulphate (SDS) is a detergent that compromises membrane stability, and as any cell wall defects increase the vulnerability of the plasma membrane to SDS, sensitivity can indicate problems with the cell wall (Bickle, Delley, Schmidt, & Hall, 1998; Igual, Johnson, & Johnston, 1996; Shimizu, Yoda, & Yamasaki, 1994). CR, Congo Red (CR) is an additional cell wall stress reagent (Wood & Fulcher, 1983). We supplemented CM culture media with Calcofluor White (200µg/ml CFW), Congo Red (200µg/ml CR), or sodium dodecyl sulphate (0.01% SDS) prior to inoculation with WT and mutant strains. Quantification of the growth inhibition rate, based on colony size, showed that the ΔMoxyl1B strain was more sensitive to cell wall stress reagents than ΔMoxyl1A, suggesting a possible role for this gene in cell wall integrity. Interestingly, we observed that double gene deletion, however, rescued the MoXYL1B phenotype to approximate that of the MoXYL1A single mutant, suggesting that the absence of MoXYL1A improves stress tolerance of the 1B mutant in M. oryzae (Figure 3). From these observations, we speculated that MoXYL1A and MoXYL1B possibly modulates stress homeostasis in M. oryzae by counter regulating either expression, or enzymatic activities of each other.
MoXYL1A and MoXYL1B localize to the cytoplasm localization in M. oryzae
The subcellular localization of the MoXYL1A and MoXYL1B proteins in M. oryzae was investigated by transforming GFP fusion constructs of both proteins under their respective native promoters into the protoplast of the Guy11 strain (Dr. Didier Tharreau, CIRAD, Montpellier, France). The cultured strains harboring the florescence signals were observed with a Nikon laser confocal and laser excitation epifluorescence microscope, showing that both fusion proteins were mainly localized in the cytoplasm during vegetative and infectious development of the rice blast fungus (Figure 4a-b). However, there was a weak GFP signal observed in conidia and the appressorium for MoXYL1B (Figure 4b). To assess expression dynamics of these genes, the transcript levels of MoXYL1A and MoXYL1B were measured during host-plant interaction at varying intervals of infection. 6-week-old rice seedlings were infected with a spore suspension of WT M. oryzae and RNA was extracted from the infected plants at 12h, 24h, 36h, 72h and 96h post inoculation for qRT-PCR assessment of MoXYL1A and MoXYL1B. Results showed that both MoXYL1A and MoXYL1B were not expressed at the hyphal stage, since control mycelia did not have detectable transcripts and we infer therefore that the genes are expressed below the limit of detection. In early infection stages, the expression of MoXYL1A and MoXYL1B was down-regulated, suggesting that these genes do not play any key role in initiation of the infection cycle (Figure 4c). However, MoXYL1A expression was significantly upregulated at 72 hpi, suggesting that MoXYL1A has some regulatory role in the later infection stages of the disease cycle. The expression profile of MoXYL1B was not highly dynamic, suggesting that it is unlikely to play a major role in the infection process and may instead have some other regulatory roles in the fungus independent of pathogenicity.
Magnaporthe oryzae mediates blast infection using appressorium-like structures produced on hyphal-tips (Kong et al., 2013; Yin et al., 2016). As noted earlier, the MoXYL1 genes were annotated as non-expressed xylanases in a prior study (Nguyen et al., 2011), which we posit was due to their potential secretion. To assess host localization of this effector protein, mycelial plugs from M. oryzae expressing MoXYL1A-GFP under its native promoter were used to inoculate barley plants and observed under a confocal microscope at different stages of disease development. Barley leaf sheath was peeled off to see the localization of the effector protein in host leaf cells. As fungal disease progressed through early stages, the invasive hyphae displayed GFP signal, and the effector protein was secreted out of hyphae at 72 hpi (Figure 5a). At this time, the barley leaf was examined to track the translocation of effector proteins within the host, at which point it was trafficked to the chloroplast (Figure 5b). The same chloroplast localization was observed upon inoculation with spore suspension in place of mycelia.
Furthermore, we endeavored to verify the localization of MoXYL1A to rice chloroplasts. An Agrobacterium tumefaciens-based MoXYL1A-GFP construct driven by the CaMV35s promoter was generated and transiently co-expressed with the rice chloroplast marker protein ChCPN10C-RFP, in Nicotiana benthamiana. Using confocal microscopy to assess protein localization, at 48 hpi MoXYL1A-GFP and Ch-CPN10C-RFP were found to be co-localized in transfected tobacco cells, confirming the localization to the chloroplast of the effector protein (Figure 6a). To ascertain the role of the chloroplast transit peptide in the chloroplast localization of the effector protein, we constructed GFP vectors with MoXYL1A lacking its chloroplast transit sequence (cTP) and co-expressed MoXYL1A-Dctp-GFP with Os-CH-RFP (a rice chloroplast marker protein) in tobacco plants. The deletion of the 42-amino acid cTP from MoXYL1A-GFP resulted in no observable GFP signal, confirming the requirement of the transit peptide for proper localization (Figure 6b). In contrast, bioinformatic tools predicted MoXYL1B to be a non-organelle targeting protein, and our tobacco infection results with MoXYL1B-GFP confirmed that it does not target any specific host organelle but instead localizes to the perifery of the host cells (Figure 6c).