Novel function of the PsDMAP1 protein in regulating the growth and pathogenicity of Phytophthora sojae.

The DNA methyltransferase 1-associated protein (DMAP1) was initially identified as an activator of DNA methyltransferase 1 (DNMT1), a conserved eukaryotic enzyme involved in diverse molecular processes, including histone acetylation and chromatin remodeling. However, the roles and regulatory mechanisms of DMAP1 in filamentous pathogens are still largely unknown. Here, employing bioinformatic analysis, we identified PsDMAP1 in P. sojae, which features a canonical histone tail-binding domain, as the ortholog of the human DMAP1. A phylogenetic analysis of DMAP1 protein sequences across diverse eukaryotic organisms revealed the remarkable conservation and distinctiveness of oomycete DMAP1 orthologs. Homozygous knockout of PsDMAP1 resulted in the mortality of P. sojae. Furthermore, silencing of PsDMAP1 caused a pronounced reduction in mycelial growth, production of sporangia and zoospore, cystospore germination, and virulence. PsDMAP1 also played a crucial role in the response of P. sojae to reactive oxygen species (ROS) and osmotic stresses. Moreover, PsDMAP1 interacted with DNA N6-methyladenine (6 mA) methyltransferase PsDAMT1, thereby enhancing its catalytic activity and effectively regulating 6 mA abundance in P. sojae. Our findings reveal the functional importance of PsDAMP1 in the development and infection of P. sojae, and this marks the initial exploration of the novel 6 mA regulator PsDMAP1 in plant pathogens.

Characterization of PcSTT3B as a Key Oligosaccharyltransferase Subunit Involved in N-glycosylation and Its Role in Development and Pathogenicity of Phytophthora capsici.

Asparagine (Asn, N)-linked glycosylation is a conserved process and an essential post-translational modification that occurs on the NXT/S motif of the nascent polypeptides in endoplasmic reticulum (ER). The mechanism of N-glycosylation and biological functions of key catalytic enzymes involved in this process are rarely documented for oomycetes. In this study, an N-glycosylation inhibitor tunicamycin (TM) hampered the mycelial growth, sporangial release, and zoospore production of Phytophthora capsici, indicating that N-glycosylation was crucial for oomycete growth development. Among the key catalytic enzymes involved in N-glycosylation, the PcSTT3B gene was characterized by its functions in P. capsici. As a core subunit of the oligosaccharyltransferase (OST) complex, the staurosporine and temperature sensive 3B (STT3B) subunit were critical for the catalytic activity of OST. The PcSTT3B gene has catalytic activity and is highly conservative in P. capsici. By using a CRISPR/Cas9-mediated gene replacement system to delete the PcSTT3B gene, the transformants impaired mycelial growth, sporangial release, zoospore production, and virulence. The PcSTT3B-deleted transformants were more sensitive to an ER stress inducer TM and display low glycoprotein content in the mycelia, suggesting that PcSTT3B was associated with ER stress responses and N-glycosylation. Therefore, PcSTT3B was involved in the development, pathogenicity, and N-glycosylation of P. capsici.

Functional Analysis of the C-5 Sterol Desaturase PcErg3 in the Sterol Auxotrophic Oomycete Pathogen Phytophthora capsici.

Although sterols play an important role in most eukaryotes, some oomycetes, including Phytophthora spp., have lost the sterol synthesis pathway. Nevertheless, the ERG3 gene encoding C-5 sterol desaturase in the sterol synthesis pathway is still present in the genomes of Phytophthora spp. Phytophthora capsici, a destructive pathogen with a broad range of plant hosts, poses a significant threat to the production of agriculture. This study focused on the ERG3 gene in P. capsici (PcERG3) and explored its function in this pathogen. It showed that the PcERG3 gene could be expressed in all tested developmental stages of P. capsici, with sporangium and mycelium displaying higher expression levels. A potential substrate of Erg3 (stellasterol) was used to treat the P. capsici wild-type strain and a PcERG3Δ transformant, and their sterol profiles were determined by GC-MS. The wild-type strain could convert stellasterol into the down-stream product while the transformant could not, indicating that PcErg3 retains the C-5 sterol desaturase activity. By comparing the biological characteristics of different strains, it was found that PcERG3 is not important for the development of P. capsici. The pathogenicity of the PcERG3Δ transformants and the wild-type strain was comparable, suggesting that PcERG3 is not necessary for the interaction between P. capsici and its hosts. Further investigations revealed that the PcERG3Δ transformants and the wild-type strain displayed a similar level of tolerance to external adversities such as unsuitable temperatures, high osmotic pressures, and intemperate pH, signifying that PcERG3 is not essential for P. capsici to cope with these environmental stresses.

Cytochrome P450 and Glutathione S-Transferase Confer Metabolic Resistance to SYP-14288 and Multi-Drug Resistance in Rhizoctonia solani.

SYP-14288 is a fungicide as an uncoupler of oxidative phosphorylation, which is effective in controlling fungal pathogens like Rhizoctonia solani. To determine whether R. solani can develop SYP-14288 resistance and possibly multi-drug resistance (MDR), an SYP-14288-resistant mutant of R. solani X19-7 was generated from wild-type strain X19, and the mechanism of resistance was studied through metabolic and genetic assays. From metabolites of R. solani treated with SYP-14288, three compounds including M1, M2, and M3 were identified according to UPLC-MS/MS analysis, and M1 accumulated faster than M2 and M3 in X19-7. When X19-7 was treated by glutathione-S-transferase (GST) inhibitor diethyl maleate (DEM) and SYP-14288 together, or by DEM plus one of tested fungicides that have different modes of action, a synergistic activity of resistance occurred, implying that GSTs promoted metabolic resistance against SYP-14288 and therefore led to MDR. By comparing RNA sequences between X19-7 and X19, six cytochrome P450s (P450s) and two GST genes were selected as a target, which showed a higher expression in X19-7 than X19 both before and after the exposure to SYP-14288. Furthermore, heterologous expression of P450 and GST genes in yeast was conducted to confirm genes involved in metabolic resistance. In results, the P450 gene AG1IA_05136 and GST gene AG1IA_07383 were related to fungal resistance to multiple fungicides including SYP-14288, fluazinam, chlorothalonil, and difenoconazole. It was the first report that metabolic resistance of R. solani to uncouplers was associated with P450 and GST genes.

The consensus Nglyco -X-S/T motif and a previously unknown Nglyco -N-linked glycosylation are necessary for growth and pathogenicity of Phytophthora.

Asparagine (Asn, N)-linked glycosylation within Nglyco -X-S/T; X ≠ P motif is a ubiquitously distributed post-translational modification that participates in diverse cellular processes. In this work, N-glycosylation inhibitor was shown to prevent Phytophthora sojae growth, suggesting that N-glycosylation is necessary for oomycete development. We conducted a glycoproteomic analysis of P. sojae to identify and map N-glycosylated proteins and to quantify differentially expressed glycoproteins associated with mycelia, asexual cyst, and sexual oospore developmental stages. A total of 355 N-glycosylated proteins was found, containing 496 glycosites, potentially involved in glycan degradation, carbon metabolism, glycolysis, or other metabolic pathways. Through PNGase F deglycosylation assays and site-directed mutagenesis of a GPI transamidase protein (GPI16) upregulated in cysts and a heat shock protein 70 (HSP70) upregulated in oospores, we demonstrated that both proteins were N-glycosylated and that the Nglyco -N motif is a target site for asparagine - oligosaccharide linkage. Glycosite mutations of Asn 94 Nglyco -X-S/T in the GPI16 led to impaired cyst germination and pathogenicity, while mutation of the previously unknown Asn 270 Nglyco -N motif in HSP70 led to decreased oospore production. In addition to providing a map of the oomycete N-glycoproteome, this work confirms that P. sojae has evolved multiple N-glycosylation motifs essential for growth.

Identification of differentially activated pathways in Phytophthora sojae at the mycelial, cyst, and oospore stages by TMT-based quantitative proteomics analysis.

Phytophthora sojae is a widely distributed, destructive oomycete plant pathogen that has been developed as a model for oomycete biology. Given the important but limited reports on the comparison of the sexual and asexual stages in Phytophthora, we performed a large-scale quantitative proteomics study on two key asexual life stages of P. sojae-the mycelium and cyst-as well as on the oospore, which is a key sexual stage. Over 29,631 peptides from 4688 proteins were analyzed. Briefly, 445 proteins, 624 proteins, and 579 proteins were defined as differentially quantified proteins in cyst vs mycelium, oospore vs cyst, and oospore vs mycelium comparisons, respectively (|log2 fold change| > 1 and P < 0.05). Compared to the mycelium and oospore, fatty acid and nitrogen metabolism were specifically induced in cysts. In oospores, the up-regulated proteins focused on RNA transport and protein processing in endoplasmic reticulum, indicating translation, folding, and the secretion of core cellular or stage-specific proteins active in oospores, which might be used for oospore germination. The data presented expand our knowledge of pathways specifically linked to asexual and sexual stages of this pathogen. BIOLOGICAL SIGNIFICANCE: The sexual spores (oospores) in oomycetes have thick cell walls and can survive in the soil for years, thus providing a primary source and allowing the reinfection of their host plant in subsequent growing seasons. However, the proteomic study on oospores remains very limited as they are generally considered to be dormant. In the present study, we successfully isolated oospores, and performed a large-scale comparative quantitative proteomics study on this key sexual stage and two representative asexual stages of P. sojae. The results provide an improved understanding of P. sojae biology and suggest potential metabolic targets for disease control at the three different developmental stages in oomycetes.