Abstract
Pyricularia oryzae (synonym Magnaporthe oryzae) is a plant pathogen causing major yield losses in cultivated rice and wheat. The P-type ATPases play important roles in cellular processes of fungi, plants, and animals via transporting specific substrates through ATP hydrolysis. Here, we characterized the roles of a P5-ATPase, Spf1, in the development and virulence of P. oryzae. Deletion of SPF1 led to decreased hyphal growth and conidiation, delayed spore germination and appressorium formation, reduced penetration and invasive hyphal extension, and attenuated virulence. Appressorium turgor, however, was not affected by deletion of SPF1. The co-localization of Spf1-GFP and an endoplasmic reticulum (ER) marker protein, Lhs1-DsRed2, indicated that Spf1 is an ER-localized P5-ATPase. An ER stress factor, 0.5 μg/ml tunicamycin (TUNI), inhibited the growth of ∆spf1, but another ER stress factor, 5 mM dithiothreitol (DTT), promoted the growth of ∆spf1. Treatment with chemicals for oxidative stress (5 mM H2O2 and 0.8 mM paraquat) also promoted the growth of ∆spf1. Gene expression assays showed that unfolded protein response (UPR) components KAR2, OST1, PMT1, ERV29, PDI1, SCJ1, SEC61, a Ca2+ channel-related P-type ATPase gene PMR1, and a calcineurin-dependent transcription factor CRZ1 were significantly up-regulated in ∆spf1, suggesting activation of UPR in the mutant. These lines of experimental evidence indicate that SPF1 is involved in some basal ER mechanisms of P. oryzae including UPR pathway and responses to ER related stresses, therefore, affecting fungal development and virulence. However, the detailed mechanism between Spf1 and virulence still awaits future researches.
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This work was funded by the National Natural Science Foundation of China (Grants 31671975 and 31871908).
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JL, FL, and YQ contributed to the study conception and design. Material preparation, data collection and analysis were performed by YQ, JW, BD, and XL. The manuscript was written by YQ, JL, and FL. All authors read and approved the final manuscript.
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294_2019_1030_MOESM3_ESM.tif
Supplementary material 3: Fig. S1 Knockout and complementation of SPF1 in Pyricularia oryzae strain 70-15. (a) Knockout strategy. Up-F/Up-R and Dn-F/Dn-R are two primer sets used to clone two flanking fragments of SPF1. The L_R/HPH_CKR primer set was used to clone the recombinational DNA fragment in null mutants. The S-F/S-R primer set was used to clone a part fragment of SPF1 in transformants. (b) Knockout events were confirmed at the DNA level. ∆spf1 showed a band of 500 bp (for β-TUBULIN, as a positive control), while the wild type had a band of 389 bp for SPF1 (upper panel). ∆spf1 had a ~ 2 kb length recombinational DNA band, while the wild type did not (lower panel). (c) Complementation of ∆spf1 by native SPF1. The expression of SPF1 in the complementation strain spf1c was confirmed in an mRNA level by RT-PCR. β-TUBULIN was used as a control. (TIFF 1805 kb)
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Supplementary material 4: Fig. S2 Spore morphology and appressorium turgor assays of Pyricularia oryzae strains. (a) Percentage of one-cell, two-cell, or three-cell conidia. (b) Appressorium turgor assays. Collapsed appressoria rates (%) of wild type, ∆spf1, and spf1c under 0.5, 1.0, 2.0, and 3.0 M glycerol solution were counted. More than 200 appressoria were counted for each strain in triple repeats. Error bars represent standard deviation. No significant differences in ∆spf1, compared with the wild type, were found by Tukey’s HSD test. (TIFF 556 kb)
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Supplementary material 5: Fig. S3 Virulence assays of the wild type, ∆spf1, and spf1c by dropping conidial suspensions onto barley leaves. (a) Penetration rate (%) of Pyricularia oryzae appressoria at 36 hpi. (b) Disease lesions on barley leaves at 72 hpi and 96 hpi. Fifteen μl of conidium suspensions (1 × 105 conidia/ml, 5 × 104 conidia/ml, or 1 × 104 conidia/ml) were dropped on 7-day-old barley leaves and incubated at 25℃. Virulence reduction of ∆spf1 was more obvious in lower concentration of spores (1 × 104 conidia/ml) than in higher spore concentrations. (TIFF 8596 kb)
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Supplementary material 6: Fig. S4 Responses of ∆spf1 to cell wall stresses. (a) Mycelial colonies of the wild type, ∆spf1, and spf1c cultured on MM media containing 50 μg/ml CFW, 25 μg/ml Congo red, or 0.0025% SDS at 28℃ under 24 h of dark conditions for 10 days. (b) Relative growth (%) of mycelial colonies on 50 μg/ml CFW, 25 μg/ml Congo red and 0.0025% SDS. The growth of ∆spf1 was increased under cell wall stresses. (TIFF 5765 kb)
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Qu, Y., Wang, J., Zhu, X. et al. The P5-type ATPase Spf1 is required for development and virulence of the rice blast fungus Pyricularia oryzae. Curr Genet 66, 385–395 (2020). https://doi.org/10.1007/s00294-019-01030-5
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DOI: https://doi.org/10.1007/s00294-019-01030-5