Plants interfere with non-self recognition of a phytopathogenic fungus via proline accumulation to facilitate mycovirus transmission

Abstract

Non-self recognition is a fundamental aspect of life, serving as a crucial mechanism for mitigating proliferation of molecular parasites within fungal populations. However, studies investigating the potential interference of plants with fungal non-self recognition mechanisms are limited. Here, we demonstrate a pronounced increase in the efficiency of horizontal mycovirus transmission between vegetatively incompatible Sclerotinia sclerotiorum strains in planta as compared to in vitro. This increased efficiency is associated with elevated proline concentration in plants following S. sclerotiorum infection. This surge in proline levels attenuates the non-self recognition reaction among fungi by inhibition of cell death, thereby facilitating mycovirus transmission. Furthermore, our field experiments reveal that the combined deployment of hypovirulent S. sclerotiorum strains harboring hypovirulence-associated mycoviruses (HAVs) together with exogenous proline confers substantial protection to oilseed rape plants against virulent S. sclerotiorum. This unprecedented discovery illuminates a novel pathway by which plants can counteract S. sclerotiorum infection, leveraging the weakening of fungal non-self recognition and promotion of HAVs spread. These promising insights provide an avenue to explore for developing innovative biological control strategies aimed at mitigating fungal diseases in plants by enhancing the efficacy of horizontal HAV transmission.

Fig. 1. Plant enhances mycovirus transmission between VIC S. sclerotiorum strains.

A Colony morphology of S. sclerotiorum strains Ep-1PN (EP), Ep-1PNA367 (A367), 1980m, SG9, and SCH941A1 (941A1) at 7 dpi (upper panel), and co-cultures of the HAVs-infected hypovirulent strain Ep-1PN as donor and the other four HAVs-free virulent strains as recipients for 5 dpi (lower panel) on PDA at 20 °C. Ep-1PN is compatible with its single-ascospore derivative Ep-1PNA367, and incompatible with the other three strains. B Virulence assay of Ep-1PNA367, 1980m, SG9, and SCH941A1 (upper panel), and their co-inoculations with Ep-1PN (lower panel) on living oilseed rape plants (72 hpi, 20 °C). The white bar represents 1 cm. C Diameters of lesions caused by Ep-1PN, Ep-1PNA367, 1980m, SG9, and SCH941A1 alone, and following co-inoculation of those strains with Ep-1PN on living oilseed rape plants (72 hpi, 20 °C), n = 76 biologically independent samples. D Analysis of diameters of lesions caused by co-inoculation of Ep-1PN with either Ep-1PNA367 or 1980m using the Bland-Altman method, illustrating that only 6.8% (5/74) of lesion size values were outside the 95% consistency limit (95% limits of agreement) of the maximum error range. E Horizontal transmission efficiency of two mycoviruses, SsDRV and SsRVL, from Ep-1PN to 1980m on PDA, living oilseed rape plants, and oilseed rape leaves previously stored in −80 °C (n = 3 and N = 40; n = 3 indicates that we conducted three independent biological repeats and N = 40 indicates that we performed 40 co-cultures for each biological repeat, with each recipient strain from the 40 co-cultures sub-cultured on plates containing hygromycin or neomycin and used to measure mycovirus transmission efficiency). Data are presented as mean ± SD, two-tailed Student’s t test. ****p < 0.0001. Source data are provided as a Source Data file.

Fig. 2. Plant suppresses expression of fungal G protein and vic-related genes upon S. sclerotiorum infection.

A Expression cluster analysis of S. sclerotiorum genes encoding G protein subunits in 1980 and Ep-1PN co-cultures in vitro and in planta. B Expression levels of genes encoding G protein subunits in 1980 alone, and 1980 and Ep-1PN co-culture in vitro and in planta. Data are presented as mean ± SD, two-tailed Student’s t test. n = 3 biologically repetitions. C Expression cluster analysis of S. sclerotiorum genes encoding proteins containing HET conserved domains in 1980 and Ep-1PN co-cultures in vitro and in planta. The relative expression of selected genes was plotted based on the threshold of RPKM value in RNA-seq data (Bioproject Accession: PRJNA908905). Red and blue indicate respectively high and low expression levels. Source data are provided as a Source Data file.

Fig. 3. Plant-derived proline increases upon S. sclerotiorum infection and promotes mycovirus transmission.

A Proline content of oilseed rape plants inoculated by HAVs-free virulent strain Ep-1PNA367 (A367) and HAVs-infected hypovirulent strain Ep-1PN (EP) at 24 hpi, and of Ep-1PNA367 and Ep-1PN mycelia (n = 15). B Proline content of A. thaliana Col-0 wildtype and p5cs2 mutant inoculated by Ep-1PNA367 at 24 hpi (n = 11). C Virulence assay of strains 1980m, Ep-1PN, and Ep-1PN and 1980m co-inoculation (Ep & 1980m) on A. thaliana Col-0 wildtype and p5cs2 mutant, and virulence assay of Ep & 1980m co-inoculation on the p5cs2 mutant following spraying with exogenous proline (36 hpi, 20 °C) (n = 14). The lesions indicated by white arrows in the upper panel was enlarged in the lower panel. D Diameters of lesions caused by Ep-1PN and 1980m co-culture on A. thaliana Col-0 wildtype, p5cs2 mutant, and p5cs2 mutant plus proline (36 hpi, 20 °C) (n = 11). E Diameters of lesions caused by 1980m with or without proline on oilseed rape (72 hpi, 20 °C), illustrating that proline addition does not induce plant resistance to S. sclerotiorum infection (n = 57). F Transmission efficiency of two mycoviruses, SsDRV and SsRVL, from Ep-1PN to 1980m on A. thaliana Col-0 wildtype and p5cs2 mutant (n = 3 for independent experiments, N = 8 for co-cultures). G Transmission efficiency of two mycoviruses, SsDRV and SsRVL, from Ep-1PN to 1980m on PDA containing proline at concentrations ranging from 3.2 µM to 320 mM (n = 3 for independent experiments, N = 12 for co-cultures). H Co-culture of Ep-1PN with 1980m on PDA (left) and PDA + P (right) (n = 16). I Upper panel, colony morphology of virus-free virulent SG9 (recipient) and six virus-infected hypovirulent strains SZ-150, SX276, Ep-1PNA367T1, WF-1, and AH98 (donors) on PDA or PDA + P for 7 days at 20 °C and corresponding mycovirus transmission efficiency (n = 24). The hypovirulent strains were infected by diverse mycoviruses, including +ssRNA mycoviruses (SsDRV, SsRVL, SsEV1, and SsHV1), dsRNA mycoviruses (SsPV1 and SsMYRV4), and −ssRNA mycoviruses (SsNSRV1), as indicated below each fungal colony. Lower panel, co-culture model diagram of SG9 and mycovirus-infected (VI) hypovirulent strains on PDA (left). Strain SG9 was co-cultured with six hypovirulent strains on PDA or PDA + P, and the associated mycovirus transmission efficiency is shown below for each fungal strain (n = 24). The transmission efficiency of all mycoviruses was increased on PDA + P. Data are presented as mean values ± SD, two-tailed Student’s t test, n represents biologically independent samples, *p < 0.05; ***p < 0.001; ****p < 0.0001; ns not significant. Source data are provided as a Source Data file.

Fig. 4. Proline alleviates the fungal non-self recognition reaction by inhibiting vic-related gene expression and ROS accumulation.

A Evans blue staining of two incompatible strains 1980m and SCH733 co-cultured on PDA and PDA + P, for the detection of the necrotic zone and PCD response at their interface. The interface region indicated by the white frame in the upper panel was enlarged in the lower panel. Scale bar, 3 mm. B TEM visualization of cell fusion between Ep-1PNA367G and 1980m on PDA and PDA + P. The fusion pore is indicated by white arrows. The vacuole formation and organelle degradation are indicated by asterisks, and cell wall thickening is indicated by black arrows. Scale bar, 1 µm. C PI staining and visualization using confocal microscopy for detection of the cell death at the interface between 1980m and Ep-1PNA367G on PDA and PDA + P. The white box indicates regions where apoptosis occurs in the two incompatible strains stained by PI, while the addition of proline significantly reduces the extent of apoptosis in these regions. Scale bar, 50 µm. D Ep-1PNA367G and 1980m interface on PDA and PDA + P visualized under confocal microscopy; the orientation of the arrows indicates that the hyphae originally labeled with one fluorescent group showed a signal of another fluorescent group, suggesting the fusion of hyphae carrying different fluorescent labels. Scale bar, 50 µm. E Volcano plot showing the number of DEGs in co-cultured VIC strains on PDA and PDA + P. F GO enrichment analysis of down-regulated DEGs from (E). The pathways marked by magenta are closely related to PCD or ROS. G Heatmap showing the expression levels of vic-related genes when two VIC strains interface on PDA, PDA + P, and oilseed rape plants. H Expression levels of genes related to G proteins and ROS in strain 1980 alone, and co-cultured with Ep-1PNA367 on PDA and PDA + P, as shown by RT-qPCR. Data are mean ± SD of n = 3 independent experiments. I NBT staining of 1980m treated with supernatant from Ep-1PNA367G. Scale bar, 0.5 cm. J Transmission efficiency of SsDRV and SsRVL from Ep-1PN to 1980m or deletion transformants (n = 24 for co-cultures). Source data are provided as a Source Data file.

Fig. 5. Proline enhances the biological efficacy of HAVs against Sclerotinia stem rot of oilseed rape and improves yield.

A Symptoms of Sclerotinia stem rot on oilseed rape after spraying with hyphal fragment suspensions of Ep-1PN with and without proline. In the upper panel, grayish-yellow colors signify diseased plants; in the lower panel, grayish-yellow discoloration of stems is evident and caused by S. sclerotiorum. B Application of Ep-1PN with and without proline improved yield and suppressed stem rot in field experiments as compared to controls (proline and water). C The putative model for plant-derived proline-mediated inhibition of fungal non-self recognition reaction to promote mycovirus transmission. The interaction between plants and phytopathogenic fungi results in increasing proline accumulation, which promotes mycovirus transmission between incompatible fungi via inhibition of fungal non-self recognition system. Donor strain Ep-1PN is incompatible with recipient strain 1980. Source data are provided as a Source Data file.