Identification of the Novel Effector RsIA_NP8 in Rhizoctonia solani AG1 IA That Induces Cell Death and Triggers Defense Responses in Non-Host Plants

Rhizoctonia solani AG1 IA is a necrotrophic fungus that causes rice sheath blight, one of the most significant rice diseases in the world. However, little is known about the pathogenic mechanisms and functions of effectors in R. solani AG1 IA. We performed functional studies on effectors in R. solani AG1 IA and found that, of 11 putative effectors tested, only RsIA_NP8 caused necrosis in the leaves of Nicotiana benthamiana. The predicted signal peptide of this protein was required to induce cell death, whereas predicted N-glycosylation sites were not required. RsIA_NP8 was upregulated during early infection, and the encoded protein was secreted. Furthermore, the ability of RsIA_NP8 to trigger cell death in N. benthamiana depended on suppressor of G2 allele of Skp1 (SGT1) and heat shock protein 90 (HSP90), but not on Mla12 resistance (RAR1) and somatic embryogenesis receptor-like kinase (SERK3). A natural variation that prevents the triggering of cell death in N. benthamiana was found in RsIA_NP8 in 25 R. solani AG1 IA strains. It is important to note that RsIA_NP8 induced the immune response in N. benthamiana leaves. Collectively, these results show that RsIA_NP8 is a possible effector that plays a key role in R. solani AG1 IA–host interactions.


INTRODUCTION
The basidiomycete Rhizoctonia solani is a necrotrophic fungal pathogen that causes disease in many crops, such as rice, wheat, corn, cotton, and soybean. It contains 14 anastomosis groups (AG1-AG13 and AGBI) (Dölfors et al., 2019). R. solani does not produce asexual spores and can survive in the soil in sclerotial form , which is a major cause of R. solani infestation. R. solani AG1 IA, the most destructive group of pathogens, causes rice sheath blight in rice-growing areas worldwide (Gautam et al., 2003;Lee and Rush, 1983). This pathogen mainly infects the leaf blade and sheath of rice plants, but all rice organs can be colonized by mycelia. Rice yield loss to R. solani AG1 IA has been recorded as high as 50% under favorable conditions (Bernardes-De-Assis et al., 2009). Despite these significant losses, little is known about the pathogenic mechanisms of R. solani AG1 IA.
Plant fungal pathogens are divided into biotrophs, hemibiotrophs, and necrotrophs based on lifestyle. Unlike biotrophs, which absorb nutrients from the cells and tissue of living hosts for colonization and growth, necrotrophs kill host cells and take nutrients from dead plant tissue (Schulmeyer and Yahr, 2017). However, all of these pathogens have a characteristic in common-effectors, which play crucial roles in promoting pathogen infection and suppressing host defenses (Koeck et al., 2011;Kabbage et al., 2015;Anderson et al., 2017). Although effectors are important, only a small proportion of the many proteins secreted by pathogens have been identified as effectors. SsCP1, an effector secreted by Sclerotinia sclerotiorum, interacts with pathogenesis-related protein 1 (PR-1) and plays an important role in successful host infection (Yang et al., 2018). The effectors SnToxA and SnTox3 recognize PR-1-PR-5 and PR-1-1, respectively, and play a crucial role in Parastagonospora nodorum-host interactions (Lu et al., 2014;Breen et al., 2016). Several effectors have been studied in species of R. solani, such as AGLIP1 , RsLysM (Dölfors et al., 2019), and RSAG8_06778 (Anderson et al., 2017), in R. solani AG1 IA R. solani AG2-2IIIB, and R. solani AG8, respectively. AGLIP1 encodes a protein of 302 amino acids (aa) to trigger non-host and host cell death and affect the host immune response .
Plant immune response depends on interactions between receptor proteins and effectors, and many nucleotide bindingleucine rich repeat (NBS-LRR) proteins have been identified as plant receptors (Stergiopoulos and de Wit, 2009). For example, the rice NBS-LRR protein-encoded gene Pi-ta interacts directly with the effector AvrPita in Magnaporthe oryzae to activate the immune response (Jia et al., 2000). Furthermore, the receptor-like proteins Cf-4, Cf-2, Cf-9, and Cf-4E in tomato interact with the effectors Avr4, Avr2, Avr9, and Avr4E in Cladosporium fulvum, respectively ( Van der Hoorn et al., 2000;Luderer et al., 2002). In addition, recognition between receptor proteins and effectors leads to host cell death, which may promote necrotrophic pathogenesis (McDonald and Solomon, 2018). QCR8, a subunit of the cytochrome b-c1 complex of the mitochondrial respiratory chain, interacts with the effector SsSSVP1 in S. sclerotiorum to produce significant plant cell death, facilitating pathogen infection (Lyu et al., 2016).
According to genome sequencing, R. solani AG1 IA encodes 965 secreted proteins, some of which may be effectors (Zheng et al., 2013). However, only a few effector genes of R. solani AG1 IA have been functionally characterized and trigger defense signaling in plants. In this study, using transient expression assay, we detected the ability of 11 putative effectors in R. solani AG1 IA to induce cell death. The effector AG1 IA_05500 (named RsIA_NP8) triggered cell death in Nicotiana benthamiana. The predicted secretion signal peptide (SP) of this effector played a key role in inducing cell death. Furthermore, we found a natural variation in RsIA_NP8 that prevented the triggering of cell death in N. benthamiana. In addition, RsIA_NP8 triggered defense responses in N. benthamiana. These results facilitate understanding of molecular mechanisms behind host-R. solani AG1 IA interactions.

RsIA_NP8 in R. solani Induces Cell Death in N. benthamiana
In previous studies, we obtained a 36.94 Mb draft genome sequence of R. solani AG1 IA and predicted 965 potential secreted proteins (Zheng et al., 2013). To detect potential novel effectors, we selected 11 genes for further testing, which are small secreted proteins (<310 aa), had a N-terminal SP, lacked a transmembrane domain, and were predicted to be effectors on http://effectorp.csiro.au/ (Sperschneider et al., 2018). The 11 putative effectors were cloned into 35S-PMDC32 expression vector to investigate their ability to induce cell death through agrobacterium-mediated transient expression in N. benthamiana leaves. Only AG1 IA-05500 (named RsIA_NP8) triggered cell death in N. benthamiana leaves 4 days after inoculation. However, a negative control of the green fluorescent protein (GFP) construct did not trigger cell death (Figure 1).

RsIA_NP8 Is Highly Conserved in Different Fungi
RsIA_NP8 encodes a 304-aa protein that contains a predicted N-terminal SP (22 aa) and two internal repeat motifs (RPT; 25-92 and 108-174 aa, respectively). In general, the effector proteins of plant pathogenic fungi are highly conserved (Lee and Rose, 2012). We found several homolog genes of RsIA_NP8 in plant pathogenic fungi that contained R. solani AG1 IB, R. solani AG1 IC, and R. solani AG3 (Supplementary Figure S1). Furthermore, we obtained homolog sequences of RsIA-NP8 from R. solani AG1 IB (RsIB_NP8) and R. solani AG1 IC (RsIC_NP8) by polymerase chain reaction (PCR) using primers designed based on R. solani AG1 IB and R. solani AG1 IC (Supplementary Table S1). Both RsIB_NP8 and RsIC_NP8 triggered cell death in N. benthamiana leaves (Figure 2A). Western blotting showed that RsIB_NP8 and RsIC_NP8 were expressed in the infiltrated N. benthamiana leaves ( Figure 2B). These results show that homologs of RsIA_NP8 are widely present in different plant pathogenic fungi, in particular three R. solani AG1 fungi.

Functional Validation of the Predicted SP of RsIA_NP8
A yeast secretion assay was used to identify the predicted SP of RsIA_NP8 (Lee and Rose, 2012). The predicted SP nucleotide sequence of RsIA_NP8 was fused in frame with the truncated pSUC2 gene, which encodes invertase lacking its own SP. The fusion vector was transformed into yeast strain YTK12, which is deficient in invertase secretion. Invertase containing functional SP degrades raffinose into simple sugars, and thus YTK12 will grow on media with raffinose as a carbon source (Oh et al., 2009;Tian et al., 2011). YTK12 strains with the predicted SP of RsIA_NP8 or the secretion signal of Phytophthora sojae Avr1b (positive control) grew on the raffinose-containing YPRAA ( Figure 3A). However, as a negative control, the N-terminus of Mg87 in M. oryzae did not grow on YPRAA medium ( Figure 3A; Gu et al., 2011). Additionally, when the SP of INF1 elicitin was substituted by that of RsIA_NP8, the fusion protein could successfully induce cell death in N. benthamiana ( Figure 3B). These results indicate that the predicted SP of RsIA_NP8 leads to the secretion of invertase and functionally secreted proteins.
RsIA_NP8 Expression During R. solani AG1 IA Infection Fungal effector genes are generally induced by transcription in invading plant cells (Stergiopoulos and de Wit, 2009). RsIA_NP8 expression is upregulated at the 24-h inoculation point (hpi) according to transcriptome data (Zheng et al., 2013). To detect change in RsIA_NP8 expression during R. solani AG1 IA infection, we studied RsIA_NP8 expression in rice cultivar 9311, which is susceptible to R. solani AG1 IA. RsIA_NP8 expression at the 12, 24, 36, 48, and 60 hpi was identified by quantitative real-time reverse transcription (qRT) PCR. RsIA_NP8 was upregulated at 24 hpi, consistent with transcriptome data (Figure 4). These findings indicate that RsIA_NP8 is upregulated during the early stage of AG1 IA infection and plays crucial roles in R. solani AG1 IA-rice interactions.

The Predicted SP and Motifs of RsIA_NP8 Are Required to Induce Cell Death
The SPs of multiple pathogenic fungi effectors are required to induce cell death in plants (Saitoh et al., 2012). To identify the function of the predicted SP of RsIA_NP8 in inducing cell death, we performed transient expression assays of RsIA_NP8 with and without the SP in N. benthamiana leaves. RsIA_NP8 sp (RsIA_NP8 lacking the SP) no longer induced cell death ( Figure 5A). Western blotting showed that RsIA_NP8 sp was expressed in the infiltrated leaves ( Figure 5B). These results show that the SP is required for RsIA_NP8 to trigger cell death in N. benthamiana. Furthermore, to clarify the function of the two predicted RPT motifs of RsIA_NP8 in inducing cell death, we analyzed deletion mutants of RsIA_NP8 in N. benthamiana using agroinfiltration. The lack of any one RPT motif did not lead to cell death ( Figure 5A). Immunoblot analyses showed that all deletion mutants of RsIA_NP8 were expressed in N. benthamiana leaves ( Figure 5B). These data indicate that the two RPT motifs are required to induce cell death.
Some effector proteins of filamentous fungal pathogens are predicted to be N-glycosylated, such as effector Slp1 in M. oryzae, which causes rice blast (Chen et al., 2014). In addition, three putative N-glycosylation sites (Asn-92, Asn-129, and Asn-271) are predicted in the RsIA_NP8 sequence. To determine whether these N-glycosylation sites are necessary for RsIA_NP8 to induce cell death, we replaced them with alanine. Cell death in N. benthamiana leaves was detected 4 days after infiltration of an agrobacterium containing the RsIA_NP8 mutation ( Figure 5C). The expression of RsIA_NP8 truncated variant (Asn-92, Asn-129, and Asn-271) in N. benthamiana was verified by Western blotting (Figure 5D). These findings indicate that the predicted N-glycosylation sites do not affect the ability of RsIA_NP8 to induce cell death.
RsIA_NP8-Triggered Cell Death in N. benthamiana Depends on SGT1 and HSP90 but Not RAR1 and SERK3/Bak1 Suppressor of G2 allele of Skp1 (SGT1), heat shock protein 90 (HSP90), and Mla12 resistance (RAR1) play crucial roles in resistance induced by R protein (Shirasu and SchulzeLefert, 2003;Shirasu, 2008). Therefore, we performed a VIGS assay against these genes in N. benthamiana to identify the function of SGT1, FIGURE 2 | Homolog sequences of RsIA_NP8 from R. solani AG1 IB (RsIB_NP8) and R. solani AG1 IC (RsIC_NP8) induce cell death in N. benthamiana leaves. (A) RsIA_NP8, RsIB_NP8, and RsIC_NP8 induced cell death in N. benthamiana. Trypan blue staining of N. benthamiana leaves, GFP was used as a negative control. BAX was used as a positive control. Typical symptoms were photographed at four dpi. Numbers e.g., 28/30, indicate that 28 of 30 infiltrated leaves exhibiting cell-death or mottling phenotypes. (B) Transient expression of RsIA_NP8, RsIB_NP8, RsIC_NP8, BAX, and GFP in N. benthamiana was confirmed by Western blotting.
HSP90, and RAR1 in cell death induced by RsIA_NP8; INF1 was used as a positive control. RsIA_NP8 induced cell death in RAR1silenced plants but not in SGT1-and HSP90-silenced plants ( Figure 6A). The expression of these genes was verified by RTquantitative PCR and was markedly reduced in silenced plants compared to the control ( Figure 6B). These results indicate that RsIA_NP8-triggered cell death depends on SGT1 and HSP90 but not RAR1. In addition, somatic embryogenesis receptor-like kinase two (SERK3)/Bak1, which encodes receptor-like kinase, is a key regulator in pathogen-associated molecular pattern (PAMP), such as programmed cell death by INF1 (Heese et al., 2007). To further determine whether RsIA_NP8 is involved in PAMP-triggered immunity (PTI) responses, we next tested whether SERK3/Bak1 mediates RsIA_NP8-induced cell death in N. benthamiana. The results showed that RsIA_NP8 induced cell death in SERK3/Bak1-silenced plants. Therefore, RsIA_NP8 is not involved in PTI responses dependent on SERK3/Bak1 in N. benthamiana.

Natural Variation Prevents RsIA_NP8 From Triggering Cell Death
We further analyzed sequences of RsIA_NP8 in 25 R. solani AG1 IA strains isolated from different areas in China. Among the 25 strains, four natural variations in RsIA_NP8 were identified with PCR (Supplementary Figure S2). To determine the ability of these variations to induce cell death, we performed transient expression assays of the RsIA-NP8 variations in N. benthamiana leaves. RsIA_NP8-20 (variation found in 20 aa) no longer induced cell death ( Figure 7A); however, cell death was induced in N. benthamiana leaves by the other variations ( Figure 7A). Western blotting showed that RsIA_NP8-20 was expressed in FIGURE 3 | The signal peptide (SP) of RsIA_NP8 is functional. (A) Functional validation of the SP of RsIA_NP8 using yeast invertase secretion assay. All transformed YTK12 yeast strains grew on YPRAA media with raffinose as the sole carbon source (1% yeast extract, 2% peptone, 2% raffinose, and 2 µg antimycin A per liter). N-terminal sequences of Phytophthora sojae Avr1b and Magnaporthe oryzae Mg87 were used as positive and negative controls, respectively. The untransformed YTK12 did not grow on either CMD-W (0.67% yeast N base without amino acids, 0.075% tryptophan dropout supplement, 2% sucrose, 0.1% glucose, and 2% agar) or YPRAA media. Yeast growth on CMD-W media was equally viable among the transformed strains.  the infiltrated leaves ( Figure 7B). These results show that this natural variation prevents RsIA_NP8 from triggering cell death in N. benthamiana.

Subcellular Localization of RsIA_NP8
To explore the subcellular localization of RsIA_NP8, we transiently expressed 2 × 35S:RsIA_NP8-YFP in tobacco leaf epidermis cells. Transient expression of 2 × 35S:YFP was used as a control. The RsIA_NP8-YFP protein was localized predominantly in the chloroplasts of the transiently transformed tobacco leaf epidermis cells compared to the control (Figure 8).

RsIA_NP8 Triggers Plant Immunity Responses
To identify whether RsIA_NP8 activates the immune response in N. benthamiana, we used qRT-PCR to evaluate the expression of several genes related to activation of the immune response in N. benthamiana. Transcription factor ERF1 (ethylene response factor 1), LOX (lipoxygenase), RbohB (respiratory burst oxidase homolog protein B), and the two pathogenesis-related (PR) protein genes PR2b and PR4a were strongly induced by RsIA_NP8 at 24 h ( Figure 9A; Wang et al., 2000;Lorenzo et al., 2003;Lee et al., 2013); however, the expression of these five genes did not strongly induce upregulation by RsIA_NP8 with SP deletion mutant at 24 h ( Figure 9A). These results indicate that RsIA_NP8 has an important role in activating early defense responses in hosts. In addition, we investigate the ability of RsIA_NP8 to activate hydrogen peroxide (H 2 O 2 ) and callose deposition in N. benthamiana leaves. It was shown that H 2 O 2 was activated by RsIA_NP8 at 4 dpi compared to the control, and extensive callose deposition was detected at 24 h after infiltration N. benthamiana leaves (Figure 9B).

DISCUSSION
Effectors play a crucial role in the successful infection of host plants by pathogens (Jones and Dangl, 2006;Stergiopoulos and de Wit, 2009). In recent years, hundreds of effectors have been predicted in various plant pathogenic fungi through genome sequencing and RNA-seq (Cantu et al., 2013;Hane et al., 2014). In the R. solani AG1 IA genome, 965 genes encode secreted proteins, of which 234 show significantly different expression during the early infection process, and 103 small cysteine-rich proteins are predicted to be effectors (Zheng et al., 2013). Li et al., 2019 reported that the effector AGLIP1 in R. solani AG1 IA induces cell death in N. benthamiana and rice protoplasts.
In this study, we selected 11 candidate effectors for functional assay; of these, only RsIA_NP8 triggered cell death in N. benthamiana. Further experiments showed that RsIA_NP8 was upregulated during the early stages of R. solani AG1 IA infection, similar to most other effectors, such as smut_2965 and smut_5844 in Tilletia horrida, which cause rice kernel smut . In addition, RsIA_NP8-triggered cell death in N. benthamiana depended on its SP. It is interesting that the effector AGLIP1 in R. solani AG1 IA also requires its predicted SP to induce cell death . This phenomenon has been observed in Ustilaginoidea virens and T. horrida (Fang et al., 2016;Wang et al., 2019). In U. virens, eight secreted proteins all required SPs to induce cell death in N. benthamiana and rice protoplasts (Fang et al., 2016). In T. horrida, the full-length effector uan2 was able to trigger cell death in N. benthamiana; however, uan2 that lacked SPs was not .These results show that these proteins have characteristics in common and might function in the extracellular space (Morgan and Kamoun, 2007). A lack of SPs means that these proteins cannot be secreted into the extracellular matrix, where proteins are recognized by plasma membrane receptors (Nie et al., 2019). Another possibility is that these secreted proteins might be translocated into cells after their secretion and recognized by cytoplasmic receptors.
The N-linked glycosylation of secreted proteins is associated with protein folding, stability, quality control, sorting, and secretion and is important for effectors Aebi, 2001, 2004). Most effector glycoproteins are dependent on N-glycosylation for more efficient secretion (Chen et al., 2014). In U. virens, mutations on the N-glycosylation sites of effectors UV_1423 and UV_6205 affected the ability to induce cell death. For UV_1423, the mutation of the predicted N-glycosylation site N49G partially prevented the induction of cell death. N39G and N53G mutations of UV_6205 had stronger effects on the inhibition of LUC activity in rice protoplasts (Fang et al., 2016). It is interesting that RsIA_NP8 was predicted by the NetNGlyc 1.0 Server to be N-glycosylated, and three N-glycosylation sites (Asn-92, Asn-129, and Asn-271) were obtained; however, mutations of these predicted N-glycosylation sites did not affect the induction of cell death. Therefore, the function of N-glycosylation of RsIA_NP8 in plant-pathogen interactions needs further study.
Effector proteins containing multiple features, some carrying RxLR motifs, are defined as RxLR effectors, and some possess repeat motifs are repeat-containing proteins (Dou and Zhou, 2012). Repeat motifs possess different types; for example, effectors of the bacterial wilt pathogen, Ralstonia solanacearum, RipAP, carry ankyrin repeats (Peeters et al., 2013), RipS1-RipS8 possess HEAT/armadillo repeats (White et al., 2009), and RipG1-RipG7 contain leucine-rich repeats (White et al., 2009). Furthermore, these repeat motifs play important role in the biological function of repeat-containing proteins effectors, such as effector localization, mediating various protein-protein interactions, and providing effector stability (Grove et al., 2008). The effector of the leaf spot pathogen of pepper and tomato, Xanthomonas euvesicatoria, XopN, possess seven tandem HEAT/armadillo-like repeats. This effector interacts with the positive regulators of host immunity, TFT1, by HEAT/armadillolike repeats (Taylor et al., 2012). In our study, RsIA_NP8 contains two new repeat motifs, and these two RPT motifs are required for its ability of induce cell death. Therefore, we suspect that these two RPT motifs play important role in the effector function of RsIA_NP8. This needs further study.
We demonstrated the ability of RsIA_NP8 to activate the N. benthamiana immune response by confirming the expression of PR genes, the activation of H 2 O 2 , and callose deposition in N. benthamiana leaves. RsIA_NP8 induced ERF1 and LOX at different inoculation times compared to the control. These two genes are related to the activity of jasmonic acid and ethylene, which play an important role in response to necrotrophic pathogens (Wang et al., 2000;Lorenzo et al., 2003). The PR genes PR2b and PR4a were also triggered in N. benthamiana leaves following transient expression of RsIA_NP8, which shows that these two PR genes might be involved in resistance to R. solani. Hydrogen peroxide and callose deposition has an important role in resistance to plant disease (Mehdy, 1994;Wu et al., 1997;Petersen et al., 2011). In our study, RsIA_NP8 triggered the activation of H 2 O 2 and callose deposition in N. benthamiana leaves. Thus, RsIA_NP8 activated the N. benthamiana immune system.
In the study, we identified the secreted protein RsIA_NP8 in the necrotrophic fungus R. solani, likely an effector, and showed that it induces cell death or defense responses in N. benthamiana leaves. However, the precise molecular mechanisms of how RsIA_NP8 participates in rice-R. solani interactions remain to be studied further.

Strains, Plant Materials, and Growth Conditions
A total of 25 R. solani AG1 IA strains were isolated from different areas in China and cultured in PDA medium (200 g potato, 20 g sucrose, 15 g agar, and 1,000 mL distilled water) at 28 • C. Agrobacterium tumefaciens GV3101 was grown on YEP medium (10 g yeast extract, 10 g 1% tryptone, 5 g NaCl, and 1,000 mL distilled water). The yeast strain YTK12 was cultivated in YPDA medium (10 g yeast extract, 20 g peptone, 20 g glucose, 20 g agar, 0.03 g adenine hemisulfate, and 1,000 mL distilled water). Antibiotics and their concentrations were as follows: kanamycin 100 µg/mL −1 , ampicillin 100 µg/mL −1 , and rifampin 25 µg/mL −1 . Rice cultivar 9311 was provided by the Department of Rice Research Institute of Sichuan Agricultural University. Nicotiana benthamiana plants were grown at 12-h/12-h nightday photoperiods at 23 • C with 60% relative humidity.

RNA Isolation and Plasmid Construction of R. solani Putative Effector Genes
The Fungal RNA Kit (Omega, Biel, Switzerland) was used to extract total RNA from R. solani AG1 IA. cDNA was obtained with the Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland). Full-length predicted effector protein-encoding genes were amplified with TransStart FastPfu Fly DNA Polymerase (TransGen Biotech, Beijing, China). All restriction enzymes and ClonExpress enzymes (Vazyme Biotech, Nanjing, China) were used following the manufacturer's instructions. Primers of these genes were designed with CE Design version 1.03 based on our reference genome sequences and included a BamHI site and a StuI site. Primer sequences are listed in Supplementary Table S2. cDNA obtained from predicted effector genes was gel-purified with a gel purification kit (Omega) and cloned into 35S-PMDC32 expression vector.

Agrobacterium tumefaciens-Mediated Transient Expression
35S-PMDC32 expression vector carrying predicted effector genes was transformed into the A. tumefaciens strain GV3101. The bacteria were centrifuged at 5,000 × g for 5 min and then resuspended in MES buffer (200 µM acetosyringone, 10 mM MgCl 2 , and 10 mM MES [pH 5.6]). The OD600 of the bacterial suspensions was adjusted to 0.5 and incubated 3 h at room temperature in the dark. The A. tumefaciens strain carrying the construct vector was infiltrated into the leaves of N. benthamiana at the four-leaf stage. Empty 35S-PMDC32-GFP vector was used as a negative control, and 35S-PMDC32 vector carrying the Bax gene was used as a positive control. A total of 20 leaves from different plants were infiltrated for each control. We observed cell death 4 days after infiltration.

Function Validation of the SP
We used yeast secretion assay to identify the function of the predicted SP of RsIA_NP8. The SP sequence of RsIA_NP8 was amplified with specific primers by PCR (primer sequences are provided in Supplementary Table S2) and cloned into pSUC2 vectors. The pSUC2 vector carrying the SP sequence of RsIA_NP8 was transformed into the yeast strain YTK12 with the Frozen-EZ Yeast Transformation II Kit (Zymo Research, Irvine, CA, United States). Then, the yeast was grown on CMD-W plates (6.7 g yeast N base without amino acids, 0.75 g tryptophan dropout supplement, 20 g sucrose, 1 g glucose, 15 g agar, and 1,000 mL distilled water, pH 5.8) and YPRAA plates (10 g yeast extract, 20 g peptone, 20 g raffinose, 2 µg antimycin A, l5 g agar, and distilled water 1,000 mL, pH 5.8).

Site-Directed Mutagenesis
We used splicing overlap extension PCR for site-directed mutagenesis. Two DNA fragments of RsIA_NP8 were amplified from the cDNA of R. solani AG1 IA, and the PCR production was cloned into 35S-PMDC32 vectors. The primer sequences used are listed in Supplementary Table S2.

Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction
The Fungal RNA Kit (Omega, Norcross, GA, United States) and Spin Column Plant Total RNA Purification Kit (Sangon Biotech, Shanghai, China) were used to extract RNA from R. solani AG1 IA strains and N. benthamiana leaves, respectively, according to the recommended protocols. The fungal conserved gene 18S rRNA was used as an internal control for data normalization; for N. benthamiana leaves, the EF1α gene was used as an internal reference gene to determine relative expression values. cDNA was obtained with the Transcriptor First Strand cDNA Synthesis Kit (Roche). The Bio-Rad CFX96 Real-Time PCR System (Bio-Rad, Foster City, CA, United States) was used for qRT-PCR according to the manufacturer's instructions. We used the 2 − Ct algorithm to calculate the relative expression of target genes. Four biological replications were performed, and the results were consistent. The primer sequences used are listed in Supplementary Table S2.

Protein Extraction and Western Blotting
Samples of N. benthamiana leaves were obtained 2 days after infiltration and ground in liquid nitrogen. A singlestep plant active protein extraction kit (Sangon Biotech) was used to extract protein from the leaves according to the manufacturer's instructions. The protein samples were separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and then transferred to nitrocellulose membranes. The membranes were blocked for 1 h at room temperature with 5% milk in TBS-T buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.05% Tween 20) and incubated with anti-FLAG antibody (1:5,000 dilution). Then the membranes were washed three times in TBS-T buffer. The immunoblots were visualized with an eECL Western blotting kit (CWBio) and photographed on X-ray films.

Trypan Blue Staining, Oxygen Burst, and Callose Deposition Detection
We observed cell death in N. benthamiana leaves using trypan blue staining. Nicotiana benthamiana leaves were collected 4 days after infiltration and soaked with aldehyde fixative for 24 h. Boiling trypan blue solution (10% lactic acid, 10% glycerol, 10% ddH 2 O, 10% phenol, and 0.67 g trypan blue) was used to dye leaves for 24 h, and then leaves were decolorized for 3 days with 2.5 g/mL chloral hydrate solution (Fernández-Bautista et al., 2016). We observed H 2 O 2 activation by staining N. benthamiana leaves with 3,3 -diaminobenzidine as described previously (Hans et al., 1997). Callose deposition was monitored at 24 h after agroinfiltration for N. benthamiana. All experiments were repeated five times.

VIGS Assay in N. benthamiana
Agrobacterium tumefaciens GV3101 strains carrying pTRV1 and pTRV2 gene were grown in YEP media (containing appropriate antibiotics) for 36 h, centrifuged, and then suspended in infiltration medium [10 mM MgCl 2 , 10 mM MES (pH 5.7), and 200 µM acetosyringone] mixed at a 1:1 ratio with OD600 = 0.5 for each strain. We then used syringes to infiltrate the cocultures in abaxial side leaves of N. benthamiana at the four-leaf stage. We verified the silencing of NbHSP90, NbSERK3, NbRAR1, and NbSGT1 after 20 days of infiltration using qRT-PCR. Three biological replications were performed, and the results were consistent. The primer sequences used are listed in Supplementary Table S2.

Subcellular Localization
The full-length sequence of RsIA_NP8 was cloned into PHB-YFP vector and then transformed into A. tumefaciens GV3101. The transformed A. tumefaciens GV3101 infiltrated N. benthamiana leaves as described previously. Subcellular localization was detected with laser confocal fluorescence microscopy 2 days after infiltration.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/Supplementary Material.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2020.01115/full#supplementary-material FIGURE S1 | Homology of RsIA_NP8 and other fungi.