Identification and localization of a new ABCG-1 subfamily protein, MoCdr1, in M. oryzae
Kim et al. reported the presence of five ABCG-1 subfamily proteins in M. oryzae genome [16]. Here, we found that five of these proteins (MGG_13624/MoABC1, MGG_00447/MoABC2, MGG_07848, MGG_10277, MGG_07375) shared high sequence similarity, with more than 35% identity. Protein structure analysis showed that these identified proteins contain two typical ATP regions (NDBs) and two trans-membrane regions (TMDs) (Fig. 1A). Notably, MGG_07848 exhibited high sequence identity of 49.9%, 49.2%, 47.8%, 47.7% and 48.3% with CaCdr1, CaCdr4, CaCdr2, and CaCdr3 of C. albicans and ScPdr5 of S. cerevisiae, especially in conserved regions (Fig. S1),. Therefore, we designated MGG_07848 as MoCDR1 in this study.
Further, we determined the subcellular localization of GFP-tagged ABCG-1 proteins, including MoCdr1, and found that all five proteins were membrane localized. The GFP signals were appeared on the cell membrane, vacuole membrane, or other inner membranes in mycelia, spores and appressoria. This was further confirmed by co-localization with a membrane-bound marker FM4-64 (Fig. 1B-D). The membrane distribution of these proteins is consistent with the typical localization characteristics of ABC proteins.
MoCDR1 responds to metal ions and oxidative stress in M. oryzae
We employed the gene replacement strategy to generate null mutants for each of the ABCG-1 genes, including MoCDR1 (Fig. S2). There were no significant differences in colony morphology and vegetative growth rates between deletion mutants and the wild type Guy11, when cultured on complete medium (CM) (Fig. S3A). However, drug sensitivity comparison revealed that these genes were involved in the fungus tolerance to various drugs (Table S1). We then assessed the growth of the wild type and ∆Mocdr1 deletion mutants (∆Mocdr1-8, ∆Mocdr1-13 and ∆Mocdr1-18) on CM containing metal stressors (calcium chloride, CaCl2; zinc chloride, ZnCl2; cuprous sulfate, Cu2SO4) and oxidative stressor (hydrogen peroxide, H2O2). The ∆Mocdr1 mutants grew more slowly on CaCl2, ZnCl2, Cu2SO4, and H2O2 media than the wild type (67.42%, 57.16%, 27.95%, and 62.0% reduction, respectively), indicating that loss of MoCdr1 caused significantly increased sensitivity to metal ions and oxidative stress. While reintroducing the intact MoCDR1 gene into the deletion mutant restored resistance to these stress factors (Fig. 2A and B). Intriguingly, the deletion of remaining four ABCG-1 genes did not affect the membrane distribution of GFP-MoCdr1 (Fig. S3C). Thus, these ABCG-1 proteins are likely independent in their subcellular distributions and functions but may complement each other in resistance against harmful chemicals.
MoCDR1 is involved in conidiation, appressorium development and pathogenicity of M. oryzae
Despite the vegetative growth of ∆Mocdr1 being comparable to the wild type Guy11 and complemented strain Mocdr1-c (Fig. 3A and S4A), the MoCDR1 deletion mutant produced significantly fewer conidia (1.5×106 conidia per plate) compared to the wild type (3×106 conidia per plate) (Fig. 3B and C). The conidial germination of ∆Mocdr1 was normal like the wild type (Fig. S4B). Then, we compared appressorium formation between the ∆Mocdr1 mutant and the wild type. The ∆Mocdr1 showed impaired appressorium formation, with the ratio of appressorium formation being significantly lower than that in the wild type after 4 ~ 10 h of incubation, and ∆Mocdr1 produced longer germ tubes (Fig. 3D and E). Additionally, during appressorium development of ∆Mocdr1, the translocation of lipid droplets and glycogen from conidia to appressoria was delayed at 8 h post inoculation (hpi), then the degradation of lipid droplets and glycogen in appressoria was hindered at 24 hpi (Fig. 3F-I). These results demonstrate that MoCDR1 is involved in regulating the translocation and utilization of storage compounds during appressorium maturation in M. oryzae.
To investigate whether ABCG-1 protein-encoding genes influence pathogenicity, we performed pathogenicity assays on rice and barley seedlings. Compared with the wild type Guy11, the pathogenicity was significantly decreased in ΔMoabc1 mutant, while remained unchanged in ΔMoabc2 mutant (Fig. S3B). Moreover, we showed that both MGG_07375 and MGG_10277 were dispensable for the pathogenicity, whereas MoCDR1 was required for the pathogenicity of M oryzae. Notably, lesions caused by the ∆Mocdr1 mutants on both rice and barley leaves were significantly reduced in number and size compared to those caused by the wild type and complemented strains. Detached inoculation assays revealed that, ∆Mocdr1-inoculated barley leaves showed markedly fewer lesions than the wild type and complemented strains-inoculated leaves (Fig. 4A and B). These results suggest that MoCDR1 is required for the full virulence of M. oryzae. We then performed appressorial infection assays with barley leaves. At 24 hpi, ∆Mocdr1 produced fewer penetration pegs compared to the wild type. At 48 hpi, the penetration rate of appressoria was 90% in the wild type while less than 30% in the ∆Mocdr1 mutant. Furthermore, the colonization of ∆Mocdr1's invasive hyphae inside the barley tissues were significantly delayed compared with the wild type (Fig. 4C and D). We also found that MoCDR1 deletion resulted in decreased phosphorylation levels of Pmk1 and Mps1 (Fig. 4E). These results suggest that MoCDR1 gene is involved in appressorium-mediated penetration and invasive growth by influencing the MAPK signaling pathway.
Identification and structural analysis of TmCDR1 in T. mentagrophytes
To identify the homologue of MoCDR1 in M. oryzae from T. mentagrophytes, we identified a total of 45 putative ABC family genes in T. mentagrophytes from the NCBI database. CDD analysis and protein structural analysis revealed that all 45 proteins possess a typical ABC structure. Phylogenetic analysis of the protein sequences further divided these 45 proteins into seven subfamilies, among which the ABCG-1 subfamily comprises six proteins (Fig. 5A). Remarkably, a gene, GBF63800, displayed 48.9%, 50.9% and 55.2% sequence homology with MoCDR1, CaCDR1 and ScPDR5, respectively. Here, GBF63800-encoded protein was designated as TmCDR1. Additionally, the two ATP-binding domains (NBDs) and two transmembrane domains (TMDs) between TmCdr1 and its homologues MoCdr1, CaCdr1, ScPdr5, TruMdr3, FgAbc3 of Fusarium graminearum showed high sequence identity (Fig. 5B and S5). We further displayed the predictive 3D (three-dimensional) structures of TmCdr1, MoCdr1 and their homologues from AlphaFold2 and AlphaFold3 database models. The six proteins have greatly similar 3D structures, particularly the α helices of transmembrane domains (Fig. 5C). These data implys that TmCdr1, MoCdr1 and their homologues remain largely conserved in both sequence and transporter conformation.
TmCDR1 is a functional homologue of MoCDR1 in drug resistance and pathogenicity
Next we determine the subcellular distribution of TmCdr1. GFP-TmCdr1 and mCherry-TmCdr1 were introduced into the wild type strain of T. mentagrophytes. In hyphae and spores, both GFP-TmCdr1 and mCherry-TmCdr1 displayed a typical membrane localization (Fig. 6A). Given the sequence homology and similar membrane distribution between TmCdr1 and MoCdr1, we sought to investigate whether these two proteins are functionally related. For this purpose, we introduced the fusion proteins GFP-TmCdr1 and mCherry-TmCdr1 into the ∆Mocdr1 mutant to obtain the ∆Mocdr1::mCherry-TmCDR1 and ∆Mocdr1::GFP-TmCDR1 strains. Surprisingly, TmCdr1 restored the resistance of ∆Mocdr1 mutant to CaCl2, ZnCl2 and H2O2 (Fig. 6B and C). Furthermore, TmCDR1 effectively reinstated the pathogenicity of the ∆Mocdr1 mutant. The disease symptoms caused by ∆Mocdr1::mCherry-TmCDR1 and ∆Mocdr1::GFP-TmCDR1 strains were comparable to those of the wild type Guy11 and ∆Mocdr1::GFP-MoCDR1 strains (Fig. 6D). Thus, these results confirm that TmCDR1 is a functional homologue of MoCDR1.
TmCDR1 mediates multidrug resistance of T. mentagrophytes
To characterize the function of the TmCDR1 gene in T. mentagrogenes, TmCDR1 null mutant was generated using a gene replacement strategy (Fig. S6). The growth, colony morphology, and conidial germination of the ∆Tmcdr1 mutants were similar to those of the wild type ZJA-1 (Fig. 7A-C). Similar to ∆Mocdr1 mutant, the ∆Tmcdr1 mutant exhibited significantly higher sensitivity to CaCl2 than the wild type. Additionally, the ∆Tmcdr1 mutant was more sensitive to the fungicides, myclobutanil (MBT) and berberine (BBR), the major effective ingredients of the traditional Chinese medicine Coptis chinensis (Fig. 7C and D).
Furthermore, we analyzed the expression of 26 out of the 45 TmABC genes in the wild type strain of T. mentagrophytes treated with distinct drugs. Treatment with BBR at a concentration of 500 µg ml− 1 led to an upregulated expression of 9 TmABC genes while decreasing the expression of 6 TmABC genes. Similarly, clotrimazole (CMZ) treatment significantly decreased the expression of 14 TmABC genes while markedly increasing the expression of 3 TmABC genes (GBF60718, GBF64815, and TmCDR1) (Fig. S7). Notably, TmCDR1 was upregulated following treatment with both BBR or clotrimazole (Fig. 7E). These results suggest an association between the ABC family genes and drug resistance in T. mentagrophytes, with TmCDR1 mediating resistance to both BBR and clotrimazole.
TmCDR1 is required for the animal infection of T. mentagrophytes
To determine whether TmCDR1 participates in infection process of T. mentagrophytes on animal skin, the conidia of the wild type ZJA-1 and ∆Tmcdr1 were inoculated on rabbit skin. Following 10 d post-inoculation, the rabbit skin sites inoculated with ∆Tmcdr1 strain produced slightly less lesions compared to the wild type, exhibiting typical skin damages (Fig. 7F). Considering that the skin lesions on the rabbits are difficult to quantify and make accurate comparisons, we further conducted comparative transcriptomic analysis of healthy and ∆Tmcdr1 or ZJA-1 infected mouse macrophages. Compared to healthy mouse macrophages, a total of 62 disease-resistant genes exhibited altered expression in macrophages following infection with ZJA-1, with 42 up-regulated and 20 were down-regulated (p-value < 0.05, log2fold change > or < 2). In ∆Tmcdr1-infected macrophages, 39 DEGs were detected, with 21 were up-regulated and 18 were down-regulated (p-value < 0.05, log2fold change > or < 2) (Fig. 7G). Among the upregulated genes in ZJA-1-infected mouse macrophages, the expression levels of FOXP3, ASB4, and WNT2B were significantly elevated (Fig. S8). FOXP3 is a transcriptional factor crucial for the development of conventional T-cells, regulating inflammation and immune response in mammalian cells [34, 35]. ASB4 (Ankyrin repeat and suppressor of cytokine signaling box protein 4) interacts with NF-κB (nuclear factor-κB) in endothelial cells and functions in the nervous system by triggering the expression of the proopiomelanocortin gene in mice [36, 37]. WNT2B activates the NF-κB pathway and enhances the expression of downstream inflammatory cytokines in mouse macrophages [38, 39]. These important disease-resistant genes were not detected in mouse macrophages infected with ∆Tmcdr1 strain. Collectively, these results indicate that TmCDR1 is required for the full virulence of T. mentagrogenes on animals.
Transcriptome changes in M. oryzae and T. mentagrophytes induced by MoCDR1 and TmCDR1 deletions
To gain a more accurately understanding of the functional correlation between MoCDR1 and TmCDR1, we performed transcriptome analysis. Compared with the wild type Guy11 strain, the ∆Mocdr1 strain exhibited 1765 up-regulated DEGs and 1204 down-regulated DEGs (Fig. 8A). In ∆Tmcdr1 strain, a total of 535 DEGs were detected compared to the wild type ZJA-1 strain, with 324 up-regulated and 211 down-regulated DEGs (Fig. 8B). KEGG analysis revealed that the DEGs of ∆Mocdr1 were mainly enriched in 11 pathways, including ribosom biogenesis, starch and sucrose metabolism, fructose and mannose metabolism, and the MAPK signaling pathway, etc. The DEGs of ∆Tmcdr1 were involved in 16 pathways, such as the biosynthesis of secondary metabolites, amino acids, glycolysis/gluconeogenesis, peroxisome, fructose and mannose metabolism, and MAPK signaling pathway, etc. Notably, the DEGs of both ∆Mocdr1 or ∆Tmcdr1 were significantly enriched in same 5 KEGG pathways: starch and sucrose metabolism, fructose and mannose metabolism, cyanoamino acid metabolism, MAPK signaling pathway, biosynthesis of secondary metabolites, and tyrosine metabolism (Fig. 8C and D).
Importantly, MAPK signaling pathway is involved in the pathogenic processes of M oryzae and T. mentagrophytes. In the ∆Mocdr1 strain, the core genes of MAPK pathway, including the MAP kinase kinase kinase Mck1 (MGG_00883) [40], high osmoregulation signaling protein Sho1 (MGG_09125) [41], protein kinase regulator Mst50 (MGG_05199) [42], were significantly downregulated. In the ∆Tmcdr1 strain, we found that MAPK pathway-related genes, such as genes encoding glycerol-3-phosphate dehydrogenase (TMEN_4625) and GTP-binding protein (TMEN_5507), were significantly upregulated. Additionally, many DEGs related to ABC membrane proteins and other transporters, such as genes encoding drug multidrug resistance proteins (MGG_12612, MGG_07375, MGG_05044/ABC6, TMEN_6461), amino acids or glucose transporters (MGG_03341, MGG_10508, MGG_06203, TMEN_3047, TMEN_6844) and metal tolerance proteins (MGG_01862, MGG_08710, MGG_05190, TMEN_2052, TMEN_6869, TMEN_9261) showed significantly alterations in the ∆Mocdr1 and ∆Tmcdr1 mutants (Fig. 8E). These results indicate that the deletion of MoCDR1 and TmCDR1 affects membrane transport of drugs, as well as pathogenic and metabolic processes, thus leading to defects in multi-drug resistance and virulence in M.oryzae and T. mentagrophytes. These similar processes were likely explain why TmCDR1 could rescue the defects of the deletion mutant ∆Mocdr1.