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.


Introduction
The oomycetes are a class of eukaryotic microorganisms that have similar life cycles and growth habits to the filamentous fungi. Oomycotes contains many pathogens of both plants and animals [1,2], including the infamous Phytophthora infestans that was responsible for the Irish potato famine of the nineteenth century and which remains a severe threat to potato production to this day [3]. In most instances, the oomycetes differ from fungi in many ways, including their genome size, ploidy of vegetative hyphae, cell wall composition (cellulose instead of chitin), and the type of mating hormones produced [4]. Phytophthora capsici is among the top five most important plant pathogens, with a wide host range, including species from Solanaceae, Leguminosae, and Cucurbitaceae. It is estimated that the worldwide vegetable production, valued at over one billion dollars, is threatened by P. capsici alone each year [5,6]. Its life cycle is divided into sexual and asexual reproductions. In the sexual life cycle, P. capsici can produce long-lived dormant oospores, which can be used as the main source of preliminary infection in soil. P. capsici is heterothallic and thus needs two strains with different mating types for sexual reproduction. At the asexual stage

The Inhibition of TM and DTT in P. capsici
To determine the functions of N-glycosylation in P. capsici, the effects of two wellknown ER stress inducers (TM and DTT) were initially investigated on the growth and development of P. capsici. TM is an N-glycosylation inhibitor, which blocks LLO formation [13,25]. DTT is a reducing agent, which prevents the formation of disulfide bonds and forms misfolded proteins in the ER [25,26]. The mycelial growth, sporangial release and zoospore production were significantly decreased when treated with TM (0.4 µg/mL or 0.7 µg/mL) or DTT (6 mM or 8 mM), compared to the untreated control (CK, without ER stress inducers) ( Figure 1A,B,D,E). TM and DTT had no significant difference on the sporangium production ( Figure 1C). Adding TM or DTT significantly increased cyst germination rates compared to the CK ( Figure 1A,F). These results demonstrated that N-glycosylation likely played an essential role in the growth and development of P. capsici. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 3 of 16 development of P. capsici. TM is an N-glycosylation inhibitor, which blocks LLO formation [13,25]. DTT is a reducing agent, which prevents the formation of disulfide bonds and forms misfolded proteins in the ER [25,26]. The mycelial growth, sporangial release and zoospore production were significantly decreased when treated with TM (0.4 µg/mL or 0.7 µg/mL) or DTT (6 mM or 8 mM), compared to the untreated control (CK, without ER stress inducers) ( Figure 1A,B,D,E). TM and DTT had no significant difference on the sporangium production ( Figure 1C). Adding TM or DTT significantly increased cyst germination rates compared to the CK ( Figure 1A,F). These results demonstrated that N-glycosylation likely played an essential role in the growth and development of P. capsici.

Sequence and Phylogenetic Analysis of PcSTT3B
As the key catalytic subunit of OST, the sequence of the core subunit STT3s of P. capsici was characterized. The genes PcSTT3A (JGI accession: Pc118787) and PcSTT3B (JGI accession: Pc541727) were identified in the JGI database [27] in the P. capsici genome. They have a high similarity to the STT3s in other Phytophthora species. PcSTT3A and PcSTT3B encode the STT3/PMT2 superfamily domain [28] (Figure 2A). Based on Sanger sequencing with cDNA and DNA of the wild-type (WT), the PcSTT3B gene contained three introns (59, 60, and 62 bp) and a 2208 bp open reading frame (ORF) encoding a 735-amino-acid protein to correct the PcSTT3B gene model (Table S4).
Phylogenetic analysis showed that most oomycetes contained two distinct STT3 isoforms, STT3A and STT3B, which are grouped into one branch. However, fungi included only one homologous STT3 protein. The PcSTT3B was highly conserved within oomycetes ( Figure 2A). Sequence alignment (Figure 2A) suggested that PcSTT3B comprised a highly canonical WWDYG motif, which acted as the enzyme activity pocket and provided a hydrogen bond to interact with the Thr/Ser of the NXT/S motif [14,29]. The PcSTT3B protein carried the DXXK motif that contributed additional contacts to the Thr/Ser of the acceptor peptide [14]. Furthermore, we identified 11 transmembrane domains in PcSTT3B ( Figure 2B), implying it was probably a transmembrane protein.
(C) Sporangium productions of all strains under a light microscope at 10× mirror magnification. (D) Sporangial release rates of different strains. (E) Zoospore productions of different strains. (F) Cyst germination rates of different strains. Data represent the mean ± standard deviations from three biological repeats. Asterisks indicate a significant difference compared to the untreated control (CK) (*, p < 0.05; **, p < 0.01).

Sequence and Phylogenetic Analysis of PcSTT3B
As the key catalytic subunit of OST, the sequence of the core subunit STT3s of P. capsici was characterized. The genes PcSTT3A (JGI accession: Pc118787) and PcSTT3B (JGI accession: Pc541727) were identified in the JGI database [27] in the P. capsici genome. They have a high similarity to the STT3s in other Phytophthora species. PcSTT3A and PcSTT3B encode the STT3/PMT2 superfamily domain [28] (Figure 2A). Based on Sanger sequencing with cDNA and DNA of the wild-type (WT), the PcSTT3B gene contained three introns (59,60, and 62 bp) and a 2208 bp open reading frame (ORF) encoding a 735-amino-acid protein to correct the PcSTT3B gene model (Table S4).
Phylogenetic analysis showed that most oomycetes contained two distinct STT3 isoforms, STT3A and STT3B, which are grouped into one branch. However, fungi included only one homologous STT3 protein. The PcSTT3B was highly conserved within oomycetes ( Figure 2A). Sequence alignment (Figure 2A) suggested that PcSTT3B comprised a highly canonical WWDYG motif, which acted as the enzyme activity pocket and provided a hydrogen bond to interact with the Thr/Ser of the NXT/S motif [14,29]. The PcSTT3B protein carried the DXXK motif that contributed additional contacts to the Thr/Ser of the acceptor peptide [14]. Furthermore, we identified 11 transmembrane domains in PcSTT3B ( Figure 2B), implying it was probably a transmembrane protein.

Transcription Profile of the PcSTT3B Gene
To investigate the potential roles of the PcSTT3B gene, we analyzed expression levels of the PcSTT3B gene in the development and infection stages, including mycelia (MY), sporangia (SP), zoospores (ZO), germinated cysts (CY), and infection stages (0, 1.5, 3, 6, 12, 24, and 48 h after infection in the susceptible pepper leaves). The reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) showed that the PcSTT3B gene was expressed in all stages as described above. In the growth and development stages, the expression levels of the PcSTT3B gene display no significant differences in the MY, SP, ZO, or CY stages ( Figure 3A). The expression levels of the PcSTT3B gene were significantly upregulated after infection for 3, 6 and 24 h, compared to the stage of 0 h ( Figure 3B). These results indicated that the PcSTT3B gene might function prominently in the pathogenic stage of P. capsici.
Motifs and domains of STT3 proteins are in the oomycetes and fungi. (B) Transmembrane analysis of PcSTT3B using TMHMM.

Transcription Profile of the PcSTT3B Gene
To investigate the potential roles of the PcSTT3B gene, we analyzed expression of the PcSTT3B gene in the development and infection stages, including mycelia sporangia (SP), zoospores (ZO), germinated cysts (CY), and infection stages (0, 1 12, 24, and 48 h after infection in the susceptible pepper leaves). The reverse transc quantitative real-time polymerase chain reaction (RT-qPCR) showed that the Pc gene was expressed in all stages as described above. In the growth and develo stages, the expression levels of the PcSTT3B gene display no significant difference MY, SP, ZO, or CY stages ( Figure 3A). The expression levels of the PcSTT3B gen significantly upregulated after infection for 3, 6 and 24 h, compared to the stage ( Figure 3B). These results indicated that the PcSTT3B gene might function promine the pathogenic stage of P. capsici.

PcSTT3B Gene Disruption and Complementation
To investigate the biological functions of PcSTT3B in P. capsici, homology-d repair (HDR)-mediated replacement of the PcSTT3B gene with an NPTII gene w ducted in the P. capsici genome using CRISPR/Cas9 [30,31]. After G418 resistance ing, PsSTT3B-deleted transformants (B8, B146) were examined by PCR and RT-qPC ysis. The transformant, which was transformed but failed to delete the PcSTT3B ge regarded as a control strain (B-CK). The transformant CB151 was complemented b troducing PcSTT3B through the CRISPR/Cas9 mediated in situ complementation m [32] ( Figure 4A). PCR analysis indicated the presence of the exogenous NPTII ge the absence of the PcSTT3B gene in the PsSTT3B-deleted transformants ( Figure 4B) tionally, RT-qPCR analysis showed that the expression of the PcSTT3B was not d in the RT-qPCR assay, whereas the expression of the PcSTT3A gene was significan regulated in the mycelia stage of the PcSTT3B-deleted transformants compared to strain BYA5 ( Figure 4C). These results indicated that the expression patterns PcSTT3B and PcSTT3A genes might be complementary.

PcSTT3B Gene Disruption and Complementation
To investigate the biological functions of PcSTT3B in P. capsici, homology-directed repair (HDR)-mediated replacement of the PcSTT3B gene with an NPTII gene was conducted in the P. capsici genome using CRISPR/Cas9 [30,31]. After G418 resistance screening, PsSTT3B-deleted transformants (B8, B146) were examined by PCR and RT-qPCR analysis. The transformant, which was transformed but failed to delete the PcSTT3B gene, was regarded as a control strain (B-CK). The transformant CB151 was complemented by reintroducing PcSTT3B through the CRISPR/Cas9 mediated in situ complementation method [32] ( Figure 4A). PCR analysis indicated the presence of the exogenous NPTII gene and the absence of the PcSTT3B gene in the PsSTT3B-deleted transformants ( Figure 4B). Additionally, RT-qPCR analysis showed that the expression of the PcSTT3B was not detected in the RT-qPCR assay, whereas the expression of the PcSTT3A gene was significantly up-regulated in the mycelia stage of the PcSTT3B-deleted transformants compared to the WT strain BYA5 ( Figure 4C). These results indicated that the expression patterns of the PcSTT3B and PcSTT3A genes might be complementary. . The Primers F1 and R1 were used to amplify the PcSTT3 gene. Primers F2 and R2 were used to amplify the NPTII gene. (C) Detection of PcSTT3A an PcSTT3B genes of different P. capsici strains were analyzed using reverse transcription quantitativ real-time PCR (RT-qPCR). (D) Colony diameters of different P. capsici strains. (E) Colony morpho ogy of P. capsici different strains cultured on the V8 agar for 3 days in darkness. Data represent th mean ± standard deviations from three biological repeats. Asterisks indicate significant deviation from the WT strain BYA5 (*, p < 0.05; **, p < 0.01).

Biological Characteristics of the PcSTT3B-Deleted Transformants
To further investigate the roles of PcSTT3B in the growth and development of P. cap sici, biological characteristics of the BYA5, B-CK, CB151, and the three PcSTT3B-delete transformants were examined. Mycelium growth, sporangial release rate and zoospor production of the PcSTT3B-deleted transformants were significantly lower compared t the BYA5, B-CK, and CB151 strains ( Figure 5A,C,D). The BYA5, B-CK, CB151, and th PcSTT3B-deleted transformants displayed no significant differences in the sporangium production and cyst germination rates ( Figure 5A,B,E). These results indicated tha PcSTT3B was involved in the mycelial growth, sporangial release rate, and zoospore pro duction.
When P. capsici colonized pepper, the expression levels of the PcSTT3B gene wer highly upregulated to varying degrees, implying that PcSTT3B may play an importan role in the infection stage. To explore whether PcSTT3B was involved in pathogenicity lesion diameters, caused by the PcSTT3B-deleted transformants, were significantly lowe than that of the BYA5, B-CK, and CB151 strains ( Figure 6A,B). The result demonstrate that PcSTT3B was involved in the zoospores-infection of P. capsici.

Biological Characteristics of the PcSTT3B-Deleted Transformants
To further investigate the roles of PcSTT3B in the growth and development of P. capsici, biological characteristics of the BYA5, B-CK, CB151, and the three PcSTT3B-deleted transformants were examined. Mycelium growth, sporangial release rate and zoospore production of the PcSTT3B-deleted transformants were significantly lower compared to the BYA5, B-CK, and CB151 strains ( Figure 5A,C,D). The BYA5, B-CK, CB151, and the PcSTT3B-deleted transformants displayed no significant differences in the sporangium production and cyst germination rates ( Figure 5A,B,E). These results indicated that PcSTT3B was involved in the mycelial growth, sporangial release rate, and zoospore production.
When P. capsici colonized pepper, the expression levels of the PcSTT3B gene were highly upregulated to varying degrees, implying that PcSTT3B may play an important role in the infection stage. To explore whether PcSTT3B was involved in pathogenicity, lesion diameters, caused by the PcSTT3B-deleted transformants, were significantly lower than that of the BYA5, B-CK, and CB151 strains ( Figure 6A,B). The result demonstrated that PcSTT3B was involved in the zoospores-infection of P. capsici.  (B) lesion diameters on peppers. Pepper leaves were inoculated with ∼100 zoospores and assessed after 3 days. All experiments were replicated three times, each had a similar result. Asterisks indicate significant deviations from the WT strain BYA5 strain (*, p < 0.05). Data represent the mean ± standard deviations from three biological repeats.

Responses of PcSTT3B to ER Stress in P. capsici
Since the absence of N-glycosylation may disrupt ER homeostasis, two well-characterized ER stress inducers, TM and DTT, were used to test the effect of PcSTT3B on the BYA5, B-CK, CB151, and PcSTT3B-deleted transformants. BYA5, B-CK, and PcSTT3B-deleted transformants were grown on V8 agar plates supplemented with 0.5 µg/mL TM or 6 mM DTT. All strains were inhibited by TM and DTT. The PcSTT3B-deleted   Pepper leaves were inoculated with ∼100 zoospores and asses after 3 days. All experiments were replicated three times, each had a similar result. Asterisks indi significant deviations from the WT strain BYA5 strain (*, p < 0.05). Data represent the mean ± sta ard deviations from three biological repeats.

Responses of PcSTT3B to ER Stress in P. capsici
Since the absence of N-glycosylation may disrupt ER homeostasis, two well-char terized ER stress inducers, TM and DTT, were used to test the effect of PcSTT3B on BYA5, B-CK, CB151, and PcSTT3B-deleted transformants. BYA5, B-CK, and PcSTT3Bleted transformants were grown on V8 agar plates supplemented with 0.5 µg/mL TM (B) lesion diameters on peppers. Pepper leaves were inoculated with ∼100 zoospores and assessed after 3 days. All experiments were replicated three times, each had a similar result. Asterisks indicate significant deviations from the WT strain BYA5 strain (*, p < 0.05). Data represent the mean ± standard deviations from three biological repeats.

Responses of PcSTT3B to ER Stress in P. capsici
Since the absence of N-glycosylation may disrupt ER homeostasis, two well-characterized ER stress inducers, TM and DTT, were used to test the effect of PcSTT3B on the BYA5, B-CK, CB151, and PcSTT3B-deleted transformants. BYA5, B-CK, and PcSTT3B-deleted transformants were grown on V8 agar plates supplemented with 0.5 µg/mL TM or 6 mM DTT. All strains were inhibited by TM and DTT. The PcSTT3B-deleted transformants were more sensitive to TM compared to the WT, B-CK, or CB151 strains. However, there were no significant changes in the inhibition rates of all strains by adding DTT (Figure 7A,B). The PcSTT3B transcript levels increased significantly after TM treatment, instead of DTT ( Figure 7D). Therefore, these results indicated that PcSTT3B might be responsible for the ER stress tolerance caused by TM in P. capsici. transformants were more sensitive to TM compared to the WT, B-CK, or CB151 strains. However, there were no significant changes in the inhibition rates of all strains by adding DTT (Figure 7A,B). The PcSTT3B transcript levels increased significantly after TM treatment, instead of DTT ( Figure 7D). Therefore, these results indicated that PcSTT3B might be responsible for the ER stress tolerance caused by TM in P. capsici. The total proteins of mycelia were extracted and analyzed by western blotting using an anti-ConA-HRP antibody. Red arrows represent certain mannosylated protein bands that slightly reduced in the PcSTT3B-deleted transformants compared to that of the BYA5 strain. β-tubulin was used as a loading control. (D) Relative PcSTT3B gene expression levels under ER stress inducers. The value of the un-treated control (CK) was set to 1 as a reference. Asterisks indicate significant differences from CK (*, p < 0.05). Data represent the mean ± standard deviations from three biological repeats.

Disruption of PcSTT3B Abolished the Attachment of Glycoproteins in Mycelium
Because PsSTT3B is a highly conserved catalytic subunit, we were interested in investigating whether the deletion of the PcSTT3B gene affected the glycoprotein content. The lectin ConA recognizes and binds to oligomannosidic glycans and had a high affinity to both terminal α-D-mannose and α-D-glucose residues of glycoproteins [33]. Certain mannosylated protein bands (red arrow) were slightly reduced in the PcSTT3B-deleted transformants compared to that of the BYA5 strain ( Figure 7C). The results demonstrated that deletion of the PsSTT3B gene decreased the high mannose-type sugar chains in some proteins in P. capsici. The total proteins of mycelia were extracted and analyzed by western blotting using an anti-ConA-HRP antibody. Red arrows represent certain mannosylated protein bands that slightly reduced in the PcSTT3B-deleted transformants compared to that of the BYA5 strain. β-tubulin was used as a loading control. (D) Relative PcSTT3B gene expression levels under ER stress inducers. The value of the un-treated control (CK) was set to 1 as a reference. Asterisks indicate significant differences from CK (*, p < 0.05). Data represent the mean ± standard deviations from three biological repeats.

Disruption of PcSTT3B Abolished the Attachment of Glycoproteins in Mycelium
Because PsSTT3B is a highly conserved catalytic subunit, we were interested in investigating whether the deletion of the PcSTT3B gene affected the glycoprotein content. The lectin ConA recognizes and binds to oligomannosidic glycans and had a high affinity to both terminal α-D-mannose and α-D-glucose residues of glycoproteins [33]. Certain mannosylated protein bands (red arrow) were slightly reduced in the PcSTT3B-deleted transformants compared to that of the BYA5 strain ( Figure 7C). The results demonstrated that deletion of the PsSTT3B gene decreased the high mannose-type sugar chains in some proteins in P. capsici.

Discussion
N-glycosylation within the N-X-Ser/Thr (X = Pro) motif is a post-translational modification that is ubiquitous and participates in various cellular processes. In P. sojae, the N-glycosylation inhibitor at 2.5 µg/mL of TM prevents the growth of P. sojae and cyst germination [34]. In Magnaporthe oryzae, 5 µg/mL of TM significantly inhibited mycelial growth, mycelial cell length, conidiophore formation, and invasive hyphal growth in host cells [26]. Similarly, TM and DTT prevented mycelial growth, sporangial release and zoospore production of P. capsici, suggesting that N-glycosylation is necessary for oomycete development. However, TM and DTT significantly increased the cyst germination rates, which might need to be maintained within a range that was consistent with the rates before the addition of TM and DTT to result in strong pathogenicity. Therefore, high germination rates, after the addition of TM or DTT, might not necessarily indicate strong pathogenicity.
The key catalytic enzyme OST is important for N-glycosylation. The eukaryotic OST complex is highly conserved, including the active center STT3 subunit and several noncatalysis subunits [14,15,18]. However, there are differences in the STT3 proteins among species. The prokaryotic OST enzyme contains a membrane-embedded single subunit: archaeal glycosylation B (AglB) for archaea, and protein glycosylation B (PglB) for bacteria, which are homologs of STT3 [35]. Leishmania major or Trypanosoma brucei have four or three STT3 copies, LmSTT3A-D or TbSTT3A-C. In other words, these unicellular parasites lack additional, non-catalytic subunit homologs of the yeast or mammal OST complex [36,37]. These single subunit OST (ssOST) enzymes display distinct protein acceptor and LLO donor specificity [38,39]. Most oomycetes utilize two distinct STT3 isoforms, similar to plants and mammals [18,40,41]. STT3A and STT3B of most oomycetes are grouped independently into one branch on the phylogenetic tree. Fungi have only an STT3 homologous protein, as do bacteria. OST3/6 interfaces with the Sec61 or Ssh1 translocon, which exists in two yeast OST complexes, to involve in cotranslational or posttranslational glycosylation in S. cerevisiae [14,15,42]. However, the DC2/KPC2 (Homologous with OST6)-containing STT3A-OST functions cotranslationally, and the MagT1/TUSC3 (Homologous with OST3)containing STT3B-OST functions posttranslationally in the mammal [20,43]. Overall, the contributions of PcSTT3A or PcSTT3B to cotranslational or posttranslational glycosylation remain unclear and need further exploration.
The OST complex transfers the LLO from the lipid carrier phosphorylated dolichol to the NXS/T motif of a nascent polypeptide in higher eukaryotes [44,45]. The deletions of the five subunits (STT3, SWP1, WBP1, OST1, and OST2) are fatal, whereas deletions of other subunits (OST3, OST4, and OST5) reduce the complex stability and glycosylation activity in S. cerevisiae [46,47]. In A. thaliana, the AtSTT3A and AtSTT3B double knockout results in gametophytic lethality [22]. The situation is different among pathogenic fungi. STT3 can be deleted in the genome of V. dahliae, whereas it cannot be deleted in A. fumigatus [23,24]. We generated the PcSTT3B-deleted transformants in P. capsici using the CRISPR/Cas9 system. The expression level of the PcSTT3A gene significantly increased in the PcSTT3B-deleted transformants. We speculate that PcSTT3A and PcSTT3B are redundant and collaborative. In P. capsici, PcSTT3A may partially supplement the function of PcSTT3B, so as to avoid cell death.
The STT3 has a catalytic active site and is conserved in the OST complex. The AtSTT3Adeleted mutant is more sensitive to salt, while the AtSTT3B-deleted mutant has no obvious phenotype in A. thaliana [22]. The AtSTT3A is involved in stomatal development by modulating the release of active abscisic acid and auxin [48]. In A. fumigatus, repression of the AfSTT3 gene leads to severe retardation of growth and a slight defect in cell wall integrity by replacing the endogenous promoter of AfSTT3 [23]. In V. dahliae, deletion of the VdSTT3 gene results in defective growth, glycoprotein secretion, and virulence [24]. In this study, PcSTT3B was involved in mycelial growth, sporangial release, zoospore production, and virulence of P. capsici. The greater number of unreleased sporangia in the PcSTT3B-deleted transformants might have resulted in defecting zoospore production. The pathogenicity in the oomycete-plant interaction is affected by many aspects, such as mycelial growth rate, cyst germination, and formation of the infection structure [49][50][51]. Interestingly, the cyst germination rate and morphology were not affected, but the zoosporeinoculated pathogenicity was significantly reduced in the PcSTT3B-deleted transformants. We speculate that the pathogenicity of the "cyst germinated mycelia" was possibly reduced during cyst germination to invade the host. Therefore, targeting the PcSTT3B may be a promising strategy for the development of fungicides with a new mode of action.
In eukaryotes, the ER is a protein-folding processing factory. To ensure correct protein folding, the ER quality control (ERQC) recognizes the Glc 3 Man 9 GlcNAc 2 structure on the NXS/T motif of the nascent polypeptides [52]. The unfolded protein response (UPR) assists in refolding the misfolded protein to avoid the escape of misfolded or intermediate proteins from the ER. The irreparable protein is degraded through the ER-related degradation (ERAD) system to ensure that only correctly folded proteins are transported into the GA [53,54]. The repression of AfSTT3 activates the UPR in A. fumigatus [23]. PcSTT3B is required for P. capsici to cope with ER stress. Therefore, the deletion of the PcSTT3B gene might have damaged ERQC and then changes the biological phenotypes. Furthermore, the changes in the biological phenotypes of the PcSTT3B-deleted transformants are partially caused by the reduced N-glycoproteins.
In the prevention and control of Phytophthora, chemical control still plays an important role. However, due to the emergence of drug-resistant strains, there is an urgent need to explore new fungicide targets. Previous studies have reported that a novel inhibitor A378-0 in the Pharmaceuticals' HitS DNA Encoding Library (DEL) was screened and characterized by using the mitogen activated protein kinases 1 (MoMps1) in Magnaporthe oryzae as bait, which inhibits the penetration and invasive growth of appressoria cells of rice blast fungus [55]. In this study, we have demonstrated that the PcSTT3B is necessary for the development, pathogenicity, and N-glycosylation of P. capsici, and can be considered a drug target for the control of Phytophthora. PcSTT3B serves as a baiting protein for the screening of small molecule compounds with novel chemical structures and good antibacterial activity in the DEL. These compounds can be used as a precursor for further design and development of fungicide compounds, as well as compounds that can be used as broad-spectrum fungicide combinations. This study provides a foundation for the effective prevention and control of various Phytophthora diseases, offering a new strategy for future research.
Overall, this study demonstrated that N-glycosylation has an indispensable and crucial role in P. capsici. However, the functions of key catalytic enzymes within the N-glycosylation are ambiguous. Hence, we identified and characterized the functions of a novel PcSTT3B gene, encoding an STT3 subunit in the OST, and analyzed its expression profile during the P. capsici life cycle. PcSTT3B was essential for vegetative growth, sporangial release rate, zoospore production, and virulence. PcSTT3B also contributed to the ER stress response and N-glycosylation. These results would enrich our knowledge of oomycete pathogenesis and consequently provide potential fungicide targets for oomycete control.

Pathogen and Plant
The P. capsici strain BYA5 was isolated from an infected pepper from the Gansu province of China in 2011. It served as the wild-type (WT) strain for gene editing to obtain transformants. The PsSTT3B-deleted transformants (B8, B146) were conducted by homology-directed repair (HDR)-mediated replacement of the PcSTT3B gene with an NPTII gene using CRISPR/Cas9 [30,31]. The transformant B-CK, which was transformed but failed to delete the PcSTT3B gene, was included as a control strain. The transformant CB151 was complemented by reintroducing PcSTT3B through the CRISPR/Cas9 mediated in situ complementation method [32]. All strains were routinely cultured on 10% V8 agar at 25 • C in darkness [56]. A susceptible pepper cultivar (Green Peak Ox Horn) was grown in a greenhouse at 27 ± 2 • C temperature, 80% relative humidity, 16 h-light/8 hdark photoperiod.

Analysis of the PcSTT3B Protein
The PcSTT3B gene was cloned from P. capsici BYA5 with the primers listed in Table  S1. Other STT3 protein sequences from different species were acquired from the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov, accessed on 20 October 2022) database and the FungiDB database (https://fungidb.org/fungidb/app, accessed on 20 October 2022) [57]. The Mega 6.0 software was used to construct the phylogenetic tree [58]. MEME server (http://meme.nbcr.net, accessed on 20 October 2022) discovered motifs and domains in these proteins [59,60]. The prediction of transmembrane domains in the PcSTT3B protein was conducted using the TMHMM Server (http://www. cbs.dtu.dk/services/TMHMM/, accessed on 15 August 2022).

P. capsici Transformation
The gene disruption and in situ complementation methods were performed using a CRISPR/Cas9-mediated gene replacement strategy [30][31][32]. Briefly, 2 days old P. capsici mycelial mats cultured in liquid pea broth medium were rinsed with ultra-pure water and suspended in 0. V8 agar containing 50 µg/mL G418 or 0.01 µg/mL oxathiapiprolin (DuPont Crop Protection, Wilmington, DE, USA) was used to select positive transformants [30][31][32]. PCR was used to detect the PcSTT3B gene replaced by NPTII in the PcSTT3B-deleted transformants. The PcSTT3B-deleted transformants and the PcSTT3B-complemented transformants were confirmed by RT-qPCR. The PsSTT3B-undeleted transformant was used as the control (B-CK) strain. The primers used in this study were listed in Table S1.

Transcription Profile of the PcSTT3B Gene
Biological materials of P. capsici in the different development stages were collected as previously described, including mycelia (MY) from V8 agar, mycelia with sporangia (SP), zoospores (ZO), germinated cysts (CY), and infection stages (0, 1.5, 3, 6, 12, 24 and 48 h after inoculation on the pepper leaves) [61]. Total RNA was extracted from above biological materials of P. capsici using the SV Total RNA Isolation Kit (Promega, Beijing, China). RNA integrity was tested via agarose gel electrophoresis. After DNase I treatment, 1 µg of total RNA used for cDNA synthesis with the PrimeScript RT reagent Kit (Takara, Beijing, China) according to the manufacturer's instruction.
RT-qPCR was performed in a qPCRsoft 3.4 system (qTower 2.2, Analytik Jena AG, Jena, Germany) using SYBR Premix Dimer Eraser Kit (CW Biotech, Beijing, China) under the following conditions: 95 • C for 2 min, 40 cycles of 95 • C for 10 s, 60 • C for 30 s to calculate cycle threshold (Ct) values. Actin and WS21 genes were used as internal control [62]. Primers used for RT-qPCR in this study are listed in Supplemental Table S1 online. The relative expression levels of genes were calculated using the 2 −∆∆Ct method [52]. Three biological replicates, each containing three technical replicates, were performed for each sample.

Phenotypic Characterization of the PsSTT3B-Deleted Transformants
To examine mycelial growth, cultural plugs (5 mm in diameter) were transferred onto V8 agar plates and incubated for 3 days, which had three replicates. Colony diameters were measured in two perpendicular dimensions.
To evaluate sporangium production, the culture was incubated at 25 • C in darkness on V8 agar plates for 3 days, and then in a 12 h-light/12 h-dark photoperiod for 5 days as previously described [63]. A microscope with 10× mirror magnification was used to count sporangia in the entire field of vision. Sporangia were observed either released zoospores or not. The sporangial release rate = the number of released sporangia/(the number of released sporangia + the number of unreleased sporangia).
To assess zoospore production, 10 mL of water was added to the plates, and then at 4 • C for 30 min and at 25 • C for 30 min. The zoospore suspensions were collected, and the concentrations were determined using a hemocytometer.
To evaluate cyst germination, the zoospore suspensions were shaken on a vortex for 1 min to form cysts and incubated at 25 • C for 3 to 8 h until most of the WT strain germinated. Cyst germination rates were calculated by counting the germinated cysts in the 100 individuals. Cysts were considered as germinated if the germ tube was longer than the cyst diameter.
To examine pathogenicity, the zoospore suspension was obtained as described above. Zoospore concentrations of all candidate strains were adjusted to 50,000 zoospores mL −1 , and 10 µL were inoculated to the back side of pepper leaves. The inoculated leaves were incubated in chambers with 80% humidity at 25 • C in darkness. The diameter of the disease lesions was measured perpendicularly after 3 days. The symptoms were observed under ultraviolet light. Three independent experiments were conducted, and each consisted of 6 to 8 replicates. All three experiments showed similar results.

Tolerance of the PcSTT3B-Deleted Transformants to ER Stress Inducers
To evaluate P. capsici with misfunctioned PcSTT3B on the tolerance to ER stress inducers, fresh 5 × 5-mm hyphal plugs of the PcSTT3B-deleted transformants and the WT strain were inoculated on V8 agar plates amended with either 0.5 µg/mL TM or 6 mM DTT and incubated at 25 • C in darkness for 3 days with three replicates. The unamended V8 agar was used as a control. Colony diameters were measured with a fine crosshair.
The inhibition rate was calculated as: (colony diameter without ER stress inducer-colony diameter with ER stress inducer)/(colony diameter without ER stress inducer-5 mm).

Western Blotting
The total proteins of P. capsici were extracted with the YT-015 Minute Total Protein Extraction Kit (Invent, Beijing, China). Protein content was measured using the Bradford (1976) method [64]. Total proteins were subjected to 10% SDS-PAGE gel for western blotting analysis via using the peroxidase-conjugated Concanavalin A (ConA-HRP) antibody (Sigma-Aldrich, Shanghai, China). β-tubulin (Biodee, Beijing, China) was used as a control.

Data Statistics
All experiments were replicated three times, each producing similar results. The data were analyzed with the one-way analysis of variance (ANOVA) method followed by the Fisher's least significant difference (LSD) test. Significance of means separation was determined based on p < 0.05 and p < 0.01.

Conclusions and Perspective
In summary, our study demonstrated that the N-glycosylation inhibitor TM, and ER stress inducer DTT hindered the growth of mycelia, sporangial release, and zoospore production in P. capsici, suggesting that N-glycosylation plays a crucial role in oomycete development. Moreover, PcSTT3B, the core subunit of OST, was found to be involved in pathogenicity, the ER stress response, and the N-glycosylation of P. capsici. This work sheds light on the molecular and biological functions of the PcSTT3B gene in P. capsici and enriches the understanding of the functional mechanisms and regulatory pathways of eukaryotic STT3B. Importantly, our results also contribute to filling the international research gap on the functions of eukaryotic STT3B in oomycetes. Data Availability Statement: All the data generated or analyzed during this study were included in this article and its additional data files.