The Conserved Effector UvHrip1 interacts with OsHGW, and Infection of Ustilaginoidea virens Regulates Defense- and Heading Date-Related Signaling Pathway.

Ustilaginoidea virens, which causes rice false smut (RFS), is one of the most detrimental rice fungal diseases and poses a severe threat to rice production and quality. Effectors in U. virens often act as a group of essential virulence factors that play crucial roles in the interaction between host and the pathogen. Thus, the functions of individual effectors in U. virens need to be further explored. Here, we demonstrated a small secreted hypersensitive response-inducing protein (hrip), named UvHrip1, which was highly conserved in U. virens isolates. UvHrip1 was also proven to suppress necrosis-like defense symptoms in N. benthamiana induced by the oomycete elicitor INF1. The localization of UvHrip1 was mainly in the nuclei and cytoplasm via monitoring the UvHrip1-GFP fusion protein in rice cells. Furthermore, Y2H and BiFC assay demonstrated that UvHrip1 interacted with OsHGW, which is a critical regulator in heading date and grain weight signaling pathways in rice. Expression patterns of defense- and heading date-related genes, OsPR1#051 and OsMYB21, were down-regulated over U. virens infection in rice. Collectively, our data provide a theory for gaining an insight into the molecular mechanisms underlying the UvHrip1 virulence function.


Introduction
Rice false smut (RFS) caused by the ascomycetous fungus Ustilaginoidea virens (Cooke) Takah (teleomorph Villosiclava virens) is one of the most important fungal diseases in rice [1][2][3]. With heavy losses of rice production worldwide, RFS control methods have growing attention recently. U. virens infects the rice florets and forms false smut balls, which is covered by chlamydospore on the infected spikelets, thereby causing a significant yield loss of up to 50% around the world [4,5]. The false smut balls also contain a variety of mycotoxins, such as ustilaginoidins and ustiloxins. Twenty-six ustilaginoidins derivatives and seven ustiloxins have been isolated and identified so far. Previous reports indicated that these secondary metabolites inhibit the assembly of tubulin and mitosis of cells in eukaryotes, are toxic to humans and animals [6][7][8][9][10].
When pathogen and host plant come in contact with each other, several elicitors are released by the pathogen, as well as plant defense mechanisms are activated to combat the infection [11,12]. Pathogen-associated molecules pattern (PAMP) from the pathogen is recognized by the pathogen  Table S1) were used as templates for polymerase chain reaction (PCR) amplification of uvhrip1. After sequencing, the multiple alignment analysis was performed and showed the nucleotide sequences of uvhrip1 were all identical in the 20 isolates, indicating UvHrip1 is highly conserved in U. virens (Figure 1b).  Table S1) were used as templates for polymerase chain reaction (PCR) amplification of uvhrip1. After sequencing, the multiple alignment analysis was performed and showed the nucleotide sequences of uvhrip1 were all identical in the 20 isolates, indicating UvHrip1 is highly conserved in U. virens (Figure 1b).

UvHrip1 Inhibits INF1-Induced Cell Death in N. benthamiana
Testing the ability of suppressing INF1-induced cell death is a useful method to identify functional effectors [37]. To investigate whether UvHrip1 regulates plant innate immunity, Agrobacterium strains carrying UvHrip1 and INF1 were co-infiltrated into N. benthamiana leaves. UvHrip1 suppresses the INF1 triggered cell death symptom in the infiltrated leaves, while green fluorescent protein (GFP) cannot. In addition, transiently expressed the UvHrip1 without SP (UvHrip1 NSP ) could also inhibit INF1 mediated cell death in N. benthamiana leaves (Figure 2a). Furthermore, ion leakage is correlate with cell death positively. The results showed the ion leakage of the leaves significantly reduced when co-expressing either UvHrip1 or UvHrip1 NSP with INF1 in comparison with that co-expressing GFP and INF1 (Figure 2b). These data demonstrated that UvHrip1 suppresses immunity-associated responses in N. benthamiana.

UvHrip1 Inhibits INF1-Induced Cell Death in N. benthamiana
Testing the ability of suppressing INF1-induced cell death is a useful method to identify functional effectors [37]. To investigate whether UvHrip1 regulates plant innate immunity, Agrobacterium strains carrying UvHrip1 and INF1 were co-infiltrated into N. benthamiana leaves. UvHrip1 suppresses the INF1 triggered cell death symptom in the infiltrated leaves, while green fluorescent protein (GFP) cannot. In addition, transiently expressed the UvHrip1 without SP (UvHrip1 NSP ) could also inhibit INF1 mediated cell death in N. benthamiana leaves (Figure 2a). Furthermore, ion leakage is correlate with cell death positively. The results showed the ion leakage of the leaves significantly reduced when co-expressing either UvHrip1 or UvHrip1 NSP with INF1 in comparison with that co-expressing GFP and INF1 (Figure 2b). These data demonstrated that UvHrip1 suppresses immunity-associated responses in N. benthamiana.

UvHrip1 is Manly Localized in the Nuclei and Cytoplasm
To investigate the subcellular localization of UvHrip1 in planta. gfp was fused to the C-terminal of UvHrip1 and UvHrip1 NSP , respectively. The fusion proteins and GFP driven by the 35S promoter were transiently expressed in rice protoplasts via PEG-mediated transformation. The result showed that both green fluorescence of UvHrip1-GFP and UvHrip1 NSP -GFP was detected in the nuclei and cytoplasm, which exhibited a similar subcellular localization of GFP transiently expressed in the rice cells ( Figure 3).

UvHrip1 Is Manly Localized in the Nuclei and Cytoplasm
To investigate the subcellular localization of UvHrip1 in planta. gfp was fused to the C-terminal of UvHrip1 and UvHrip1 NSP , respectively. The fusion proteins and GFP driven by the 35S promoter were transiently expressed in rice protoplasts via PEG-mediated transformation. The result showed that both green fluorescence of UvHrip1-GFP and UvHrip1 NSP -GFP was detected in the nuclei and cytoplasm, which exhibited a similar subcellular localization of GFP transiently expressed in the rice cells ( Figure 3).

UvHrip1 Interacts with Heading Date-and Grain Weight-Related Protein OsHGW
To investigate the potential functional mechanism of UvHrip1 in rice, the yeast two-hybrid (Y2H) system was performed to preliminarily screen host proteins interacting with UvHrip1. A heading date-and grain weight-related protein, named OsHGW, was identified from a rice cDNA library when UvHrip1 NSP was the bait. Moreover, the interaction between full length UvHrip1 and OsHGW was further confirmed via one-to-one validation ( Figure 4a).
The in vivo interaction between UvHrip1 and OsHGW was further investigated by bimolecular fluorescence complementation (BiFC) in N. benthamiana leaves. OsHGW and UvHrip1 or UvHrip1 NSP were fused in frame with the N-terminal domain (nYFP) and C-terminal domain of yellow fluorescence protein (cYFP), respectively. When OsHGW-nYFP were co-expressed with UvHrip1-cYFP or UvHrip1 NSP -cYFP in N. benthamiana leaves, the fluorescence signal was observed in the nuclei. By contrast, no fluorescence was detected in the control ( Figure 4b).
Collectively, these results demonstrated that UvHrip1 interacts with OsHGW in vitro and in vivo.

UvHrip1 Interacts with Heading Date-and Grain Weight-Related Protein OsHGW
To investigate the potential functional mechanism of UvHrip1 in rice, the yeast two-hybrid (Y2H) system was performed to preliminarily screen host proteins interacting with UvHrip1. A heading date-and grain weight-related protein, named OsHGW, was identified from a rice cDNA library when UvHrip1 NSP was the bait. Moreover, the interaction between full length UvHrip1 and OsHGW was further confirmed via one-to-one validation (Figure 4a).
The in vivo interaction between UvHrip1 and OsHGW was further investigated by bimolecular fluorescence complementation (BiFC) in N. benthamiana leaves. OsHGW and UvHrip1 or UvHrip1 NSP were fused in frame with the N-terminal domain (nYFP) and C-terminal domain of yellow fluorescence protein (cYFP), respectively. When OsHGW-nYFP were co-expressed with UvHrip1-cYFP or UvHrip1 NSP -cYFP in N. benthamiana leaves, the fluorescence signal was observed in the nuclei. By contrast, no fluorescence was detected in the control (Figure 4b).
Collectively, these results demonstrated that UvHrip1 interacts with OsHGW in vitro and in vivo.

Expression Analysis of Defense-and Heading Date-Related Genes in Young Rice Panicles During U. virens Infection
To elucidate whether the expression patterns of defense-and heading date-related genes were regulated during U. virens infection, the strain P1, a highly virulent isolate, was artificially inoculated into young panicles of the rice cultivar LYP9 with high P1 susceptibility [36,38]. The expression level of OsPR1#051 and OsMYB21 [39] were measured at 0, 4, 8, 12 and 16 days post-inoculation by

Expression Analysis of Defense-and Heading Date-Related Genes in Young Rice Panicles during U. virens Infection
To elucidate whether the expression patterns of defense-and heading date-related genes were regulated during U. virens infection, the strain P1, a highly virulent isolate, was artificially inoculated into young panicles of the rice cultivar LYP9 with high P1 susceptibility [36,38]. The expression level of OsPR1#051 and OsMYB21 [39] were measured at 0, 4, 8, 12 and 16 days post-inoculation by quantitative real-time reverse transcription-PCR (qRT-PCR). Compared to the expression level in mock-treated plants, OsPR1#051 and OsMYB21 were transcriptionally inhibited during pathogen infection (Figure 5a,b). The result indicated that U. virens hijacks host immune response and heading date-related signaling pathway, thus facilitating infection. uvhrip1 or pGBKT-uvhrip1 nsp in yeast strain Gold. Quadruple dropout medium (QDO) is used for interaction screening. The pGADT7-T plasmid was transformed to yeast with pGBKT7-53 or pGBKT7-Lam for positive and negative controls, respectively. T, pGADT7-T; 53, pGBKT7-53; λ, pGBKT7-Lam. (b) The in planta interaction between UvHrip1 and OsHGW was indicated by bimolecular fluorescence complementation (BiFC). Green fluorescence of N. benthamiana cells was observed by confocal microscopy at 3 days post agroinfiltration. The red arrows indicated nuclei.

Expression Analysis of Defense-and Heading Date-Related Genes in Young Rice Panicles During U. virens Infection
To elucidate whether the expression patterns of defense-and heading date-related genes were regulated during U. virens infection, the strain P1, a highly virulent isolate, was artificially inoculated into young panicles of the rice cultivar LYP9 with high P1 susceptibility [36,38]. The expression level of OsPR1#051 and OsMYB21 [39] were measured at 0, 4, 8, 12 and 16 days post-inoculation by quantitative real-time reverse transcription-PCR (qRT-PCR). Compared to the expression level in mock-treated plants, OsPR1#051 and OsMYB21 were transcriptionally inhibited during pathogen infection (Figure 5a, b). The result indicated that U. virens hijacks host immune response and heading date-related signaling pathway, thus facilitating infection.

Discussion
Rice false smut, caused by U. virens, occurs at the late stage of rice development, reduces grain yield and quality. The disease has been reported in most rice-growing areas of China and emerged as one of the major diseases in rice [1,4]. Many studies have been carried out to reduce the yield loss caused by RFS. However, little is known about the molecular mechanism underlying the interaction between rice and U. virens. Phytopathogenic microbes secrete the majority of effectors to regulate plant immunity by targeting different host key components [19,40]. More than 600 secreted proteins have been predicted in U. virens genome, 193 of which are identified as candidate effectors. Many putative effectors genes were found to be transcriptionally induced during U. viren infection in rice via expression profiling analyses, suggesting they may be involved in inhibiting immunity-associated responses [2]. In this study, we demonstrated that UvHrip1 is a potential effector regulating plant defense responses during pathogen infection.
Core effector of pathogen shows a similar sequence and conserved motif across species [24,28]. BLAST searches against the EMBL-EBI database indicated UvHrip1 is a hypersensitive response-inducing protein (Hrip) elicitor, which is similar to MoHrip2 in M. oryzae [41], and highly conserved in U. virens isolates (Figure 1). Alignment analysis demonstrated that the full length of UvHrip1 and MoHrip2 showed 67% identities. The Hrip-elicitors have been identified to improve plant resistance to the pathogen, such as Hrip1 from Alternaria tenuissima [42], PaNie from Pythium aphanidermatum [43], and MoHrip1 from M. oryzae [41]. The defense responses are often accompanied by HR, ion influx, accumulation of nitric oxide (NO) and production of reactive oxygen species (ROS) [44]. However, no cell death symptoms were monitored within 3 days after UvHirp1-expressing Agrobacterium inoculated into N. benthamiana. Possibly, UvHirp1 induces cell death in the later time after Agrobacterium inoculation or perceived by specific R protein as an avirulence protein to trigger HR in the host. Therefore, the precise function of UvHirp1 will be confirmed by further experiments in rice.
A variety of effectors secreted by biotrophic and semi-biotrophic plant pathogens can suppressed cell death in plants and are required for full virulence for infection. The ability to suppress INF1-induced cell death has been used to identify many putative functional effectors employing Agrobacterium-mediated transient expression assay in N. benthamiana [2,29,30]. In this study, we demonstrated that UvHrip1 suppresses INF1 triggered cell death in N. benthamiana. Moreover, the UvHrip1 truncated without signal peptide also inhibits INF1-induced cell death (Figure 2), indicating UvHrip1 may function as a cytoplasmic effector and act inside the cell. Further investigations, such as ROS assay, callose deposition, immunization-related genes expression, will be performed to confirm the plant immunity suppressing abilities of UvHrip1. Subcellular localization detected by confocal microscopy showed that both UvHrip1-GFP and UvHrip1 NSP -GFP were mainly localized to cytoplasm and nuclei in rice protoplasts ( Figure 3). The results indicated UvHrip1 secreted by U. virens might have multiple functions in plant [45]. However, the multiple cellular site localization of UvHrip1 cannot be ruled out because the fusion constructs are overexpressed in rice protoplasts. Hence, the precise localization of the protein in plant cells needs to be further explored.
The effectors have been reported to disable the plant immune system using multiple biochemical strategies and by targeting a variety of host proteins [19]. Here, the host target of UvHrip1 was screened to gain an insight into the molecular mechanisms underlying the UvHrip1 virulence function. OsHGW was initially identified to interact with UvHrip1 through the Y2H system. The in vivo interaction of UvHrip1 and OsHGW was subsequently confirmed through BiFC in N. benthamiana leaves (Figure 4). OsHGW contains a ubiquitin-associated (UBA) domain and regulates rice heading date and grain weight; The rice mutant oshgw delays heading by 20 days and reduces grain weight, but the number of grains per main panicle and the numbers of panicle per plant were not influenced; Heading date-and grain weight-related genes were differentially regulated in the oshgw [46]. Hence, whether UvHrip1 regulates the flower-open and immune response of rice by interacting with OsHGW remains to be further investigated.
Pathogens, which successfully colonize host tissues/organs, should have the ability to hijack or evade host immunity [47]. Here, we found the expression patterns of rice defense-and heading date-related genes, OsPR1#051 and OsMYB21, were both transcriptionally inhibited over U. virens infection ( Figure 5). OsPR1#051 is homologous of PR1 in Arabidopsis, which is associated with salicylic acid (SA) signaling pathway [39]. Rice genome encodes 12 PR1 members, all of which are transcriptionally induced during compatible and/or incompatible M. oryzae strains infection [48]. Our results indicated that the SA-mediated defense response and heading date-related signaling pathways in rice spikelets might play an essential role in the interaction between rice and U. virens.
In summary, we identified and characterized a novel secreted protein UvHrip1 as a conserved effector that suppresses immunity in non-host plant and interacts with OsHGW, which is a key regulator in heading date and grain weight signaling pathways. However, the precise molecular mechanism of UvHrip1's role in the interaction between rice and U. virens remains to be further elucidated.

Plant Materials, Pathogen Strains and Growth Conditions
U. virens isolate strains were cultured using PSA medium (200 g peeled potato extract boiled in water, 20 g sucrose and 16 g agar/L). N. benthamiana was growth in an artificial climate chamber at 14 h light (25°C) / 12 h dark (23°C). Agrobacterium GV3101 and EHA105 for transient expression were cultured using LB medium (10 g tryptone, 5 g yeast extract and 10 g NaCl/L). Yeast strain Gold was cultured using YPDA medium (10 g yeast extract, 20 g peptone, 20 g glucose, 0.03 g adenine hemisulfate/L). In this study, the concentrations of antibiotics were used as follows (µg/mL): rifampin, 25; kanamycin, 50, ampicillin, 50. All data were repeated at least three times, and the results were similar. Strains and plasmids used in this study were listed in Supplementary Tables S1 and S2.

Plasmids Construction
The total RNA of U. virens was extracted using the RNA extraction kit (TaKaRa, Japan), and the concentration and quality of that were determined by NanoDrop 2000. The cDNA synthesis was performed using PrimeScript TM 1st Strand cDNA Synthesis Kit (TaKaRa, Japan). The full-length and the truncated without signal peptide of UvHrip1 coding sequence amplified by Phanta Max ultra-fidelity DNA polymerase using the cDNA as a template.
For INF1-induced cell death inhibition assay, Xma I and Sal I digested PCR products were subcloned into pGR107 [49]. For subcellular localization, the PCR products containing the coding sequence of UvHrip1 and UvHrip1 NSP , was cloned into pUC19-35S-gfp [28] after digestion with BamH I and Sal I, respectively. All recombination constructs were determined by sequencing. Primers used in this study are listed in Supplementary Table S3.

Transient Expression of Proteins in N. benthamiana Mediated by Agrobacterium
The constructed plasmid was transformed into Agrobacterium strains EHA105 and GV3101 by the freeze-thaw method [50]. The positive transformation was verified by PCR. The overnight cultured Agrobacterium carrying the correct plasmid was collected, washed 3 times with sterile double distilled water, and resuspended in 10 mM MgCl 2 buffer (containing 10 mM MES, 10 mM acetosyringone). The optical cell density was adjusted to OD600 = 0.5 for UvHrip1-or UvHrip1 NSP -containing strain; OD600 = 0.3 for INF1-containing strain. The Agrobacterium containing the corresponding plasmid was infiltrated into 4-5 weeks old N. benthamiana by needleless syringe. Results were observed and photographed after 3 days post-inoculation.

Inoculation of U. virens in Rice and qRT-PCR
Artificial inoculation was performed as described previously [20]. Briefly, P1 was cultured for 5-7 days at 120 rpm and 28°C in the dark in PS medium. Mycelia and conidia were re-mixed at a concentration of 1 × 10 6 conidia /mL with PS medium. The inocula were injected into the rice panicles by needle syringe before rice heading stage. Rice spikelets collected at 0, 4, 8, 12 and 16 days post-inoculation were stored at −80°C for subsequent experiments.
RNA extraction and cDNA synthesis were performed as described above. qRT-PCR was performed by ChamQ SYBR Color qPCR Master Mix from Vazyme Biotech Co., Ltd. and detected according to the manufacturer's instructions by the Bio-Red CFX96 system. The internal reference gene primers used for normalizing each sample were listed in Supplementary Table S3.

Ion Leakage in N. benthamiana Leaf Discs
The ion leakage assay in N. benthamiana leaf discs to evaluate cell death was as described previously [36]. Briefly, five inoculated leaf discs of 9 mm diameter were collected and incubated in 5 mL distilled water for 3 h. The conductivity of the bathing solution was measured by a conductivity meter (FE30; Mettler Toledo). Return the leaf discs to the bathing solution, boil it in a sealed tube, and measure the conductivity of the solution again. The conductivity ratio was calculated as ion leakage.

Rice Protoplast Transfection and Subcellular Localization
Rice protoplasts isolation and transfection were carried out as described previously [51]. Briefly, protoplasts were transfected with the corresponding vector via polyethylene glycol (PEG)-mediated transfection after isolating from Oryza sativa cv. Nipponbare etiolated seedlings, and then the transfected protoplasts were incubated in solution buffer under weak light for 12-16 h.
For subcellular localization, GFP fluorescence from the overnight protoplasts was monitored using confocal microscopy (Olympus FV3000).

Yeast Two-Hybrid Screening
The Matchmaker TM Gold yeast two-hybrid system (Clontech) was used for protein-protein interaction screening in this study [52,53]. The coding sequence of uvhrip1 nsp was cloned into pGBKT7 to generate bait for screening in rice cDNA. The cDNA was synthesized by OE-Biotech Co., Ltd. (Shanghai, China). For one-to-one validation, the coding sequence of uvhrip1 or uvhrip1 nsp and OsHGW were cloned into pGBKT7 and pGADT7, respectively. Preparation of yeast competent cells and transformation were performed using a Frozen-EZ Yeast Transformation II Kit™ (ZYMO Research) following the manufacturer's instructions. The constructed pGBKT7 and pGADT7 plasmids were pairwise co-transformed into the yeast strain Gold. The protein-protein interaction in yeast was analyzed on the SD double dropout (DDO, SD/-Trp-Leu) medium and SD quadruple dropout (QDO, SD/-Trp-Leu-His-Ade) medium plates.

Bimolecular Fluorescence Complementation Assays
The full-length and truncated without signal peptide of uvhrip1 were in frame fused with the 5 -end of coding sequence of YFP in pSPYNE and OsHGW was cloned into pSPYCE using the respective specific primers (Supplementary Table S3) [54]. The constructs were transformed into Agrobacterium strain EHA105 using the freeze-thaw method. Overnight-cultured Agrobacterium strains were collected and resuspended in induction medium (10 mM MES, pH = 5.6, 10 mM MgCl 2 and 150 µM acetosyringone) to a final concentration of OD600 = 0.5. After incubating at room temperature for 2 h, Agrobacterium cultures with the pSPYNE and pSYPCE constructs were co-infiltrated into leaves of 4-5 week-old N. benthamiana plants. YFP or green fluorescence in the infiltrated N. benthamiana leaves was monitored using confocal microscopy (Olympus FV3000).