Functional Analysis of Mannosyltransferase-Related Genes UvALGs in Ustilaginoidea virens

Abstract

Rice false smut, caused by Ustilaginoidea virens, is one of the three major rice diseases in China. It not only seriously affects the rice yield and quality but also endangers human and animal health. Studying the pathogenic mechanism of U. virens has important theoretical significance and application value for clarifying the infection characteristics of the pathogen and cultivating disease-resistant varieties. Plant pathogenic fungi utilize secreted effectors to suppress plant immune responses, which can function in the apoplast or within host cells and are likely glycosylated. However, the posttranslational regulation of these effectors remains unexplored. Deletion of ΔUvALG led to the cessation of secondary infection hyphae growth and a notable decrease in virulence. We observed that ΔUvALG mutants triggered a significant increase in reactive species production within host cells, akin to ALG mutants, which plays a crucial role in halting the growth of infection hyphae in the mutants. ALG functions by sequestering chitin oligosaccharides to prevent their recognition by the rice chitin elicitor, thereby inhibiting the activation of innate immune responses, including reactive species production. Our findings reveal that ALG3 possesses three N-glycosylation sites, and the simultaneous Alg-mediated N-glycosylation of each site is essential for maintaining protein stability and chitin-binding activity, both of which are critical for its effector function. These outcomes underscore the necessity of the Alg-mediated N-glycosylation of ALG to evade host innate immunity.

Keywords: Agrobacterium tumefaciens-mediated transformation; Ustilaginoidea virens; pathogenicity; target gene knockout.

Figure 1

Expression analysis of mannosyltransferase: (A) expression analysis of UvALGs in different stages of rice infected with U. virens; (B) expression analysis of UvALGs in U. virens grown on medium for different days. (* = 0.01 < p < 0.05, ** = p < 0.01, *** = p < 0.001).

Figure 2

ALGs bioinformatics analysis of U. virens: (A) UvALG32 gene was located on the second chromosome, and UvALG3 and UvALG15 were located on the third chromosome, as shown in Figure 2; (B) three mannosyltransferases have conserved domains—ALG3, GT15, and OCH1—that are consistent with homologous genes; (C) in order to further clarify the function of the three genes, the UvALG3, UvALG15, and UvALG32 proteins were compared with the mannose transferases of Fusarium graminearum and Saccharomyces cerevisiae. The results show that UvALG3 had the highest homology with S. cerevisiae ALG3 (96%), UvALG15 had the highest homology with S. cerevisiae KTR1 (100%), and UvALG32 had the highest homology with F. graminearum OCH1 (100%).

Figure 3

Construction of ALG knockout vectors of U. virens. (A) Acquisition of upstream and downstream fragments of knockout vectors: (M) 2K DNA Maker, (1) upstream fragment, (2) downstream fragment. (B) Knockout vector enzyme digestion verification: (M) 5K DNA Maker, (1) knockout vector; (2) PXEH vector. (C) PCR verification of Agrobacterium tumefaciens transformation: (M) 2K DNA Maker, (1) upstream segment, (2) HYG internal gene, (3) downstream fragment. (D) PCR verification of ∆UvALG knockout transformants: (M) 2K DNA Maker, (1) ∆UvALGs, (2) WT (wild type). (E) Upstream and downstream PCR verification of ΔUvALG knockout transformants: (1,3) WT (wild type) and (2,4) ΔUvALGs. (F) The UvALGs’ sequences were used as templates, and the upstream and downstream homologous arms of the knockout vectors were designed using Primer 5 and DNAMAN 6.0.3.48 to amplify specific primers. Combined with the pXEH vector sequence, the enzyme digestion site and homologous arm were added to the 5’ end of the specific primer. The upstream restriction sites of the UvALG knockout vectors were EcoR I and Kpn I, and the downstream restriction sites were Xba I and Hind III. The vector construction strategy was to first connect the upstream fragment and then connect the downstream fragment. The specific strategy is as shown in the figure. The flanking sequence of about 1000 bp at both ends of the target gene was amplified by PCR, and the two flanking sequences were connected to the pXEH vector.

Figure 4

Construction of ALG gene complementation vectors of U. virens. (A) Acquisition of upstream and downstream fragments of the complement vector: (M) 2K DNA Maker, (1) upstream fragment, and (2) downstream fragment. (B) Complement vector enzyme digestion verification: (M) 10K DNA Maker and (1) complement vector. (C) PCR verification of Agrobacterium tumefaciens transformation: (M) 2K DNA Maker, (1) upstream segment, (2) NPT-II internal gene, and (3) downstream fragment. (D) PCR verification of C∆UvALG knockout transformants: (M) 2K DNA Maker, (1) C ∆UvALGs, and (2) WT (wild type). (E) Construction strategy of the complementary carrier.

Figure 5

Hygromycin resistance screening and mutant acquisition in U. virens. (A) Screening of the minimum tolerance concentration of hygromycin to U. virens. Hygromycin PSA culture with different concentrations (0 µg/mL, 50 µg/mL, 100 µg/mL, 150 µg/mL, 200 µg/mL, 250 µg/mL, and 300 µg/mL) was inoculated on Aspergillus oryzae cake with a diameter of 5 mm. Three replicates were set for each concentration, and the growth of Ustilaginoidea virens at different concentrations was observed after 14 days in culture at a constant temperature of 28 °C. The results show that there were different degrees of growth at 0 µg/mL and 200 µg/mL. Finally, the tolerance of Ustilaginoidea virens to hygromycin was 250 µg/mL. (B) ΔUvALG and CΔUvALG colony morphologies. The co-transformed strains were grown on glass paper on PSA medium (300 µg/mL cephalosporin and 250 µg/mL hygromycin B) for 7 days, and single colonies were picked and subcultured on new PSA medium. After single spore isolation, the purified strains were selected from the new PSA medium to obtain three mutants of ∆UvALG3, ∆UvALG15, and ∆UvALG32 genes. Complementary vector screening replaced the hygromycin resistance fragment with G418. (C) Gene knockout and complement mutant colony diameter. The WT strain and all gene knockout and complemented mutants were cultured on PSA medium for 14 days and the colony diameter was measured. After 7 days of shaking culture in PS medium, the sporulation was calculated using a blood cell counting plate. The colony’s morphology is shown in the image. The results show that after 14 days of culture, the average colony diameter of the WT strain was 6.13 ± 0.05 cm, and the average colony diameters of knockout mutants ΔUvALG3, ΔUvALG15, and ΔUvALG32 were 4.80 ± 0.31 cm, 5.21 ± 0.29 cm, and 4.79 ± 0.21 cm, respectively. Compared with the WT strain, the growth rate was significantly reduced. (* = p < 0.05).

Figure 6

RT-PCR verification of UvALG mutants and complements. (A) Knockout target gene verification: (1.3.5) ∆UvALGs’s target gene and (2.4.6) WT (wild type). (B) Complementary target gene verification: (1.2.3) C∆UvALG 3, C∆UvALG 15, and C∆UvALG 32 and (4.5) WT (wild type). (C) Verification of the β-tubulin reference gene in Ustilaginoidea virens: (1.2.3) ∆UvALG3, ∆UvALG15, and ∆UvALG32; (4.5.6) CΔUvALG3, CΔUvALG15, CΔUvALG32, and (7) WT (wild type); and (M) 2K Maker.

Figure 7

UvALG knockout and complementation transformants were inoculated on rice. (A) Incidence of UvALG knockout and complementation transformants. The wild-type strain, WT, mannosyltransferase gene knockout and complementation transformants of Aspergillus oryzae were cultured in PSB medium for 7 days, ground into homogenate, adjusted for concentration, and inoculated with Liaoyou 65. After 21 days, the incidence was investigated and the number of rice balls was counted. The incidence is shown in the figure. (B) Pathogenicity of UvALG knockout and complementary transformants. * p < 0.05.

Figure 8

Effects of different chemical stresses on strains. (A) Growth of UvALG knockout and complementation transformants under different stress conditions. (B) For the UvALG3 and UvALG32 genes, the inhibition rate of 120 µg/mL CR PSA medium on the mycelial growth of knockout transformants was almost the same as that of wild-type strains WT and CΔUvALG3 strains. The inhibition rate of 120 µg/mL CFW PSA medium on the mycelial growth of the knockout transformants was significantly lower than that of the wild-type strains WT and CΔUvALG3 strains. Under the pressure stress of 0.4 M sorbitol and 0.25 M NaCl, the inhibition rate of mycelial growth of the knockout transformants was significantly lower than that of the wild-type strains WT and CΔUvALG3 strains. On PSA medium containing 0.015% H₂O₂ and 0.02% SDS, the mycelial growth inhibition rate of knockout transformants was higher than that of WT and CΔUvALG3. The results showed that the deletion of UvALG3 and UvALG32 genes significantly affected the sensitivity of U. virens to different osmotic pressures and cell wall integrity. Under the stress of 120 µg/mL CFW, it became more insensitive, and under the stress of 0.4 M sorbitol and 0.25 M Na Cl, it became more sensitive. The difference is that UvALG15 under the stress of 0.4 M sorbitol and 0.25 M NaCl, the mycelial growth inhibition rate of knockout transformants and the growth inhibition rate of wild-type strains WT and CΔUvALG15 strains were not significantly different.