Contribution of a WRKY Transcription Factor, ShWRKY81, to Powdery Mildew Resistance in Wild Tomato

Tomato powdery mildew, caused by Oidium neolycopersici, is a destructive fungal disease that damages almost all of the aerial parts of tomato, causing devastating losses in tomato production worldwide. WRKY transcription factors are key regulators of plant immunity, but the roles of ShWRKYs in wild tomato Solanum habrochaites LA1777 against O. neolycopersici still remain to be uncovered. Here, we show that ShWRKY81 is an important WRKY transcription factor from wild tomato Solanum habrochaites LA1777, contributing to plant resistance against O. neolycopersici. ShWRKY81 was isolated and identified to positively modulate tomato resistance against On-Lz. The transient overexpression of the ShWRKY81-GFP (green fluorescent protein) fusion protein in Nicotiana benthamiana cells revealed that ShWRKY81 was localized in the nucleus. ShWRKY81 responded differentially to abiotic and biotic stimuli, with ShWRKY81 mRNA accumulation in LA1777 seedlings upon On-Lz infection. The virus-induced gene silencing of ShWRKY81 led to host susceptibility to On-Lz in LA1777, and a loss of H2O2 formation and hypersensitive response (HR) induction. Furthermore, the transcripts of ShWRKY81 were induced by salicylic acid (SA), and ShWRKY81-silenced LA1777 seedlings displayed decreased levels of the defense hormone SA and SA-dependent PRs gene expression upon On-Lz infection. Together, these results demonstrate that ShWRKY81 acts as a positive player in tomato powdery mildew resistance.


Introduction
Oidium neolycopersici (Ol) is a destructive obligate biotroph that causes powdery mildew disease worldwide in more than 60 host species in 13 plant families, especially members of the Solanaceae family [1]. It can infect the aerial parts of susceptible tomato plants and generate typical powdery white spots on the leaf surfaces, petioles, stems, and calyx [1]. Severe infections cause leaf etiolation and premature senescence, leading to a notable reduction in fruit production and quality [2]. Hence, it is important to adopt management practices to control the disease, among which creating and cultivating resistant varieties is a promising strategy. Fortunately, wild tomato provides a potentially useful germplasm resource of significant resistance genes, which enable breeders to efficiently enhance the resistance of susceptible tomato cultivars.
Two decades ago, several Ol-resistant genes were identified and mapped in wild tomato species, such as five dominantly inherited resistance genes (Ol-1, Ol-3, Ol-4, Ol-5, and Ol-6) and one recessive gene (ol-2) [3,4]. Until recently, studies on the functional characterization of genes from resistant wild tomato Solanum habrochaites have advanced our understanding of the tomato defense against O. neolycopersici. ShARPC3 encoding an actin-related protein is induced by O. neolycopersici and confers disease resistance in tomato LA1777 [5]. ShNPSN11-silenced plants display an elevated susceptibility to O. neolycopersici, with a significant decrease in H 2 O 2 accumulation and hypersensitive

WRKY18 and WRKY81 Were Differentially Expressed in Tomato following O. neolycopersici Infection
To find the potential resistance-related components of WRKY genes in tomato, the transcripts of~25 WRKY genes were screened by quantitative real-time PCR (qRT-PCR) in both the resistant cultivar LA1777 and the susceptible cultivar Money-maker (MM) following On-Lz infection. The transcripts of two WRKY genes changed differently in LA1777 and MM following On-Lz infection. The transcripts of WRKY18 accumulated significantly in the susceptible cultivar MM (Figure 1a). The transcripts of WRKY81 accumulated markedly in the resistant cultivar LA1777 (Figure 1b), implying that WRKY18 and WRKY81 may be involved in tomato resistance to O. neolycopersici. In particular, the transcripts of WRKY81 in LA1777 were~45 and~30 times higher than that in MM at 24 and 48 hpi (hours post inoculation), respectively ( Figure 1b). Here, ShWRKY81 was chosen as a candidate resistance-related WRKY gene for further study.

WRKY18 and WRKY81 Were Differentially Expressed in Tomato following O. neolycopersici Infection
To find the potential resistance-related components of WRKY genes in tomato, the transcripts of ~25 WRKY genes were screened by quantitative real-time PCR (qRT-PCR) in both the resistant cultivar LA1777 and the susceptible cultivar Money-maker (MM) following On-Lz infection. The transcripts of two WRKY genes changed differently in LA1777 and MM following On-Lz infection. The transcripts of WRKY18 accumulated significantly in the susceptible cultivar MM (Figure 1a). The transcripts of WRKY81 accumulated markedly in the resistant cultivar LA1777 (Figure 1b), implying that WRKY18 and WRKY81 may be involved in tomato resistance to O. neolycopersici. In particular, the transcripts of WRKY81 in LA1777 were ~45 and ~30 times higher than that in MM at 24 and 48 hpi (hours post inoculation), respectively ( Figure 1b). Here, ShWRKY81 was chosen as a candidate resistance-related WRKY gene for further study.  6,12,24,48,72,96, and 120 hpi (hours post inoculation). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal reference. Values are shown as the means ± standard error of three biological replicates, and statistically significant differences in the compatible reaction and incompatible reaction at the same time point are represented by asterisks (Student's t-test, *, p < 0.05).

Characterization of ShWRKY81
The open reading frame (ORF) of ShWRKY81 was validated to be 876 bp, producing a deduced protein containing 291 amino acids with a calculated molecular weight of 33.25 kDa and an isoelectric point of 5.51. The deduced protein comprises a WRKY domain which predictably binds to the W-box (TTGACT/C) cis-element, and a nuclear localization sequence (RRGCYKRRKTS) at amino acids 88 to 98 of its N-terminus (Figure 2b), localized in the nucleus predicted by Cell-PLoc 2.0. Phylogenetic analysis showed that ShWRKY81 clusters with SlWRKY81 ( Figure 2a). Amino acid sequence alignment revealed that ShWRKY81 has a conserved C2HC zinc finger motif ( Figure 2b) and belongs to the group III WRKY subfamily. To confirm the localization of ShWRKY81, the protein fused with a green fluorescent protein was co-expressed with a nuclear marker fused with a red fluorescent protein in Nicotiana benthamiana leaves by the Agrobacterium-mediated infiltration. The green fluorescence of ShWRKY81-GFP was only observed to overlap with the red fluorescence of the nuclear marker (Figure 3), suggesting that ShWRKY81 localizes in the nucleus. To obtain more information about the possible function of ShWRKY81, the protein blast-based searches were performed using the plant transcription factor database. The results show that the deduced protein may be involved in SA-and JA-mediated signaling pathways.  6,12,24,48,72,96, and 120 hpi (hours post inoculation). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal reference. Values are shown as the means ± standard error of three biological replicates, and statistically significant differences in the compatible reaction and incompatible reaction at the same time point are represented by asterisks (Student's t-test, *, p < 0.05).

Characterization of ShWRKY81
The open reading frame (ORF) of ShWRKY81 was validated to be 876 bp, producing a deduced protein containing 291 amino acids with a calculated molecular weight of 33.25 kDa and an isoelectric point of 5.51. The deduced protein comprises a WRKY domain which predictably binds to the W-box (TTGACT/C) cis-element, and a nuclear localization sequence (RRGCYKRRKTS) at amino acids 88 to 98 of its N-terminus (Figure 2b), localized in the nucleus predicted by Cell-PLoc 2.0. Phylogenetic analysis showed that ShWRKY81 clusters with SlWRKY81 ( Figure 2a). Amino acid sequence alignment revealed that ShWRKY81 has a conserved C2HC zinc finger motif ( Figure 2b) and belongs to the group III WRKY subfamily. To confirm the localization of ShWRKY81, the protein fused with a green fluorescent protein was co-expressed with a nuclear marker fused with a red fluorescent protein in Nicotiana benthamiana leaves by the Agrobacterium-mediated infiltration. The green fluorescence of ShWRKY81-GFP was only observed to overlap with the red fluorescence of the nuclear marker (Figure 3), suggesting that ShWRKY81 localizes in the nucleus. To obtain more information about the possible function of ShWRKY81, the protein blast-based searches were performed using the plant transcription factor database. The results show that the deduced protein may be involved in SA-and JA-mediated signaling pathways.

Expression Patterns of ShWRKY81 in Response to Abiotic Stresses and Defense Signaling Hormones
To explore whether ShWRKY81 responded to diverse abiotic stresses, the transcripts of this gene were monitored in 4-week-old LA1777 seedlings treated with low temperature (10 • C), high temperature (40 • C), 200 mM NaCl, and 20% (w/v) PEG6000, respectively. For temperature treatment, the transcripts of ShWRKY81 were markedly induced by low temperature, and exhibited a peak value in accumulation observed at 6 hpt (hours post treatment),~20-fold higher than that in control seedlings (Figure 4a). The transcripts of ShWRKY81 maintained their basal level at 1-6 hpt, and then were enhanced up-regulated at 12 and 24 hpt compared with the control after high-temperature treatment (Figure 4b).
For NaCl and PEG6000 treatments, the transcripts of ShWRKY81 were dramatically decreased compared with the control in both treatments (Figure 4c,d). Taken together, these results indicate that ShWRKY81 was differentially responsive to temperature, salinity, and drought stresses.    control (35S::GFP) in combination with 35S::NLS-dsRED (nuclear marker) were transiently overexpressed in the leaf cells of N. benthamiana via Agrobacterium-mediated transformation. Green and red fluorescence were detected and captured by a laser confocal scanning microscope at 48 h post agroinfiltration. The panels from left to right show GFP images (GFP), dsRED images (DsRed), bright images (Bright), and merged images (Merged), respectively. Bar = 20 µm.

Expression Patterns of ShWRKY81 in Response to Abiotic Stresses and Defense Signaling Hormones
To explore whether ShWRKY81 responded to diverse abiotic stresses, the transcripts of this gene were monitored in 4-week-old LA1777 seedlings treated with low temperature (10°C), high temperature (40°C), 200 mM NaCl, and 20% (w/v) PEG6000, respectively. For temperature treatment, the transcripts of ShWRKY81 were markedly induced by low temperature, and exhibited a peak value in accumulation observed at 6 hpt (hours post treatment), ~20-fold higher than that in control seedlings (Figure 4a). The transcripts of ShWRKY81 maintained their basal level at 1-6 hpt, and then were enhanced up-regulated at 12 and 24 hpt compared with the control after high-temperature treatment (Figure 4b).
For NaCl and PEG6000 treatments, the transcripts of ShWRKY81 were dramatically decreased compared with the control in both treatments (Figure 4c,d). Taken together, these results indicate that ShWRKY81 was differentially responsive to temperature, salinity, and drought stresses.
To check the predicted role of ShWRKY81 involved in SA and JA signaling, the transcripts of the gene were examined in 4-week-old LA1777 seedlings with exogenous SA or JA treatment. As expected, compared with the control, the transcripts of ShWRKY81 were significantly elevated following SA or JA treatment (Figure 4e,f). In particular, in the SA treatment group, ShWRKY81 transcripts were immediately and strongly induced and maintained higher abundance with a peak value in accumulation observed at 0.5 hpt (Figure 4e). Collectively, these data suggest that ShWRKY81 may play a role in the SA and JA signaling pathways.  To check the predicted role of ShWRKY81 involved in SA and JA signaling, the transcripts of the gene were examined in 4-week-old LA1777 seedlings with exogenous SA or JA treatment. As expected, compared with the control, the transcripts of ShWRKY81 were significantly elevated following SA or JA treatment (Figure 4e,f). In particular, in the SA treatment group, ShWRKY81 transcripts were immediately and strongly induced and maintained higher abundance with a peak value in accumulation observed at 0.5 hpt ( Figure 4e). Collectively, these data suggest that ShWRKY81 may play a role in the SA and JA signaling pathways.

Silencing of ShWRKY81 Resulted in a Loss of Host Resistance to On-Lz in LA1777
To confirm the role of ShWRKY81 in LA1777 against On-Lz infection, a tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) system was used to knockdown ShWRKY81 expression in LA1777. Phytoene desaturase (PDS), the marker gene for gene silencing, was used as a positive control to test the VIGS system. TRV2, TRV2::ShPDS, and TRV2::ShWRKY81 constructs were used to infiltrate LA1777 leaves for silencing. At 4 weeks after agroinfiltration, plants infiltrated with TRV2::ShPDS construct displayed a photo-bleaching phenotype (Figure 5b), suggesting the successful induction of VIGSmediated gene silencing. At this point, all seedlings carrying TRV2 or TRV2:ShWRKY81 constructs were inoculated with On-Lz. In parallel, samples from these seedlings were collected to examine the efficiency of gene silencing by qRT-PCR. As shown in Figure 5c

Silencing of ShWRKY81 Resulted in a Loss of Host Resistance to On-Lz in LA1777
To confirm the role of ShWRKY81 in LA1777 against On-Lz infection, a tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) system was used to knockdown ShWRKY81 expression in LA1777. Phytoene desaturase (PDS), the marker gene for gene silencing, was used as a positive control to test the VIGS system. TRV2, TRV2::ShPDS, and TRV2::ShWRKY81 constructs were used to infiltrate LA1777 leaves for silencing. At 4 weeks after agroinfiltration, plants infiltrated with TRV2::ShPDS construct displayed a photo-bleaching phenotype (Figure 5b), suggesting the successful induction of VIGS-mediated gene silencing. At this point, all seedlings carrying TRV2 or TRV2:ShWRKY81 constructs were inoculated with On-Lz. In parallel, samples from these seedlings were collected to examine the efficiency of gene silencing by qRT-PCR. As shown in Figure 5c

Silencing of ShWRKY81 Decreased Defense Responses following On-Lz Infection.
To further explore the function of ShWRKY81 in LA1777 resistance to On-Lz, the early defense responses, including H2O2 accumulation and hypersensitive response (HR) induction, were investigated during the infection process at 24, 48, and 72 hpi. For histological observation, 3,3-diaminobenzidine and trypan blue staining were separately used to evaluate the generation rate of H2O2 and HR. The H2O2 accumulations were observed at the infection sites in both ShWRKY81-silenced plants and control plants, while the generation Values are shown as the means ± standard error of three biological replicates, and statistically significant differences are represented by asterisks (Student's t-test, *, p < 0.05).

Silencing of ShWRKY81 Decreased Defense Responses following On-Lz Infection
To further explore the function of ShWRKY81 in LA1777 resistance to On-Lz, the early defense responses, including H 2 O 2 accumulation and hypersensitive response (HR) induction, were investigated during the infection process at 24, 48, and 72 hpi. For histological observation, 3,3-diaminobenzidine and trypan blue staining were separately used to evaluate the generation rate of H 2 O 2 and HR. The H 2 O 2 accumulations were observed at the infection sites in both ShWRKY81-silenced plants and control plants, while the generation rates of H 2 O 2 in the control plants were dramatically higher than that in ShWRKY81silenced plants as the infection progressed (Figure 6a,b). At 72 hpi, the generation rate of H 2 O 2 reached 55.84% in the control plants, which is~2.8 times higher than that in ShWRKY81-silenced plants (Figure 6c). In the case of trypan blue staining, the inductions of HR in control seedlings were significantly higher than that in ShWRKY81-silenced plants at 48 and 72 hpi (Figure 7a-c), similar to the trend of H 2 O 2 accumulation. Taken together, these data demonstrate that a decrease in ShWRKY81 transcripts reduces the strength of resistance responses to On-Lz infection. rates of H2O2 in the control plants were dramatically higher than that in ShWRKY81-silenced plants as the infection progressed (Figure 6a,b). At 72 hpi, the generation rate of H2O2 reached 55.84% in the control plants, which is ~2.8 times higher than that in ShWRKY81-silenced plants (Figure 6c). In the case of trypan blue staining, the inductions of HR in control seedlings were significantly higher than that in ShWRKY81-silenced plants at 48 and 72 hpi (Figure 7a-c), similar to the trend of H2O2 accumulation. Taken together, these data demonstrate that a decrease in ShWRKY81 transcripts reduces the strength of resistance responses to On-Lz infection.     rates of H2O2 in the control plants were dramatically higher than that in ShWRKY81-silenced plants as the infection progressed (Figure 6a,b). At 72 hpi, the generation rate of H2O2 reached 55.84% in the control plants, which is ~2.8 times higher than that in ShWRKY81-silenced plants (Figure 6c). In the case of trypan blue staining, the inductions of HR in control seedlings were significantly higher than that in ShWRKY81-silenced plants at 48 and 72 hpi (Figure 7a-c), similar to the trend of H2O2 accumulation. Taken together, these data demonstrate that a decrease in ShWRKY81 transcripts reduces the strength of resistance responses to On-Lz infection.    Blue staining (trypan) shows the hypersensitive cell death. The HR production rates were calculated at 24, 48, and 72 hpi (c). Co, conidium; Sh, secondary hyphae; HR, hypersensitive response. Bar, 20 µM. Values are shown as the means ± standard error of three biological replicates, and statistically significant differences are represented by asterisks (Student's t-test, *, p < 0.05).

Silencing of ShWRKY81 Reduced SA Accumulation and Led to Down-Regulated SA-Dependent PRs Gene Expression following On-Lz Infection
To investigate whether the defense hormones SA and JA contribute to ShWRKY81mediated resistance to On-Lz, SA and JA levels were measured in ShWRKY81-silenced LA1777 and control seedlings at 12 and 24 hpi with On-Lz infection. In the control group, SA levels at 24 hpi were significantly increased to 182.5% of those at 12 hpi, reaching 4068.1 nmol/g (Figure 8a), while in the ShWRKY81-silenced group, SA levels were maintained at the same level at 12 and 24 hpi (Figure 8a). In addition, SA levels at 24 hpi in the control group were significantly higher than those in the ShWRKY81-silenced group (Figure 8a). In both the control and ShWRKY81-silenced group, JA levels at 12 and 24 hpi with On-Lz did not exhibit a significant difference (Figure 8d).
To investigate whether the defense hormones SA and JA contribute to ShWRKY81mediated resistance to On-Lz, SA and JA levels were measured in ShWRKY81-silenced LA1777 and control seedlings at 12 and 24 hpi with On-Lz infection. In the control group, SA levels at 24 hpi were significantly increased to 182.5% of those at 12 hpi, reaching 4068.1 nmol/g (Figure 8a), while in the ShWRKY81-silenced group, SA levels were maintained at the same level at 12 and 24 hpi (Figure 8a). In addition, SA levels at 24 hpi in the control group were significantly higher than those in the ShWRKY81-silenced group (Figure 8a). In both the control and ShWRKY81-silenced group, JA levels at 12 and 24 hpi with On-Lz did not exhibit a significant difference (Figure 8d).
To further explore the role of the SA and JA signaling pathways in ShWRKY81-mediated resistance to On-Lz, the transcripts of marker genes (ShPR1, ShPR5, and ShPDF1.2) for these two pathways were analyzed at 24, 48, and 120 hpi. Following On-Lz infection, the transcripts of ShPR1 and ShPR5 were elevated in both groups. However, the transcripts of ShPR1 and ShPR5 in the ShWRKY81-silenced group were markedly lower than those in the control group (Figure 8b,c). Additionally, there was no obvious difference in ShPDF1.2 expression between the ShWRKY81-silenced group and control group ( Figure  8e). Taken together, these data indicate that SA signaling, but not JA signaling, may partially contribute to ShWRKY81-mediated resistance to On-Lz. Values are shown as the means ± standard error of three biological replicates, and statistically significant difference are represented by asterisks (Student's t-test, *, p < 0.05).

Discussion
Tomato powdery mildew, caused by the obligate biotrophic pathogen Oidium neolycopersici, is a devastating fungal disease in tomato worldwide and seriously limits the production of tomato due to the lack of resistant commercial cultivars [1,34]. To improve the resistance of cultivated tomatoes against O. neolycopersici, the researchers focused on the resistance evaluation of wild tomato resources and the identification of resistant genes. Solanum habrochaites, one type of such wild tomato resources, was reported to exhibit high resistance to O. neolycopersici and has been used as a material for the cloning and functional analysis of candidate resistance genes [5,6,35]. Plant WRKY proteins act as key reg- To further explore the role of the SA and JA signaling pathways in ShWRKY81mediated resistance to On-Lz, the transcripts of marker genes (ShPR1, ShPR5, and Sh-PDF1.2) for these two pathways were analyzed at 24, 48, and 120 hpi. Following On-Lz infection, the transcripts of ShPR1 and ShPR5 were elevated in both groups. However, the transcripts of ShPR1 and ShPR5 in the ShWRKY81-silenced group were markedly lower than those in the control group (Figure 8b,c). Additionally, there was no obvious difference in ShPDF1.2 expression between the ShWRKY81-silenced group and control group (Figure 8e). Taken together, these data indicate that SA signaling, but not JA signaling, may partially contribute to ShWRKY81-mediated resistance to On-Lz.

Discussion
Tomato powdery mildew, caused by the obligate biotrophic pathogen Oidium neolycopersici, is a devastating fungal disease in tomato worldwide and seriously limits the production of tomato due to the lack of resistant commercial cultivars [1,34]. To improve the resistance of cultivated tomatoes against O. neolycopersici, the researchers focused on the resistance evaluation of wild tomato resources and the identification of resistant genes. Solanum habrochaites, one type of such wild tomato resources, was reported to exhibit high resistance to O. neolycopersici and has been used as a material for the cloning and functional analysis of candidate resistance genes [5,6,35]. Plant WRKY proteins act as key regulators in physiological and biological processes, responding to stimuli, and play an essential role in modulating plant immunity [9][10][11]. Herein, we identified a WRKY transcription factor ShWRKY81 in wild tomato S. habrochaites LA1777 and characterized its positive role in tomato resistance against O. neolycopersici.
Reactive oxygen species (ROS) function as major signaling molecules in plants and can be induced at invasion sites during pathogen infection [36][37][38]. The concentration of H 2 O 2 , a relatively stable form of ROS, is determined by host resistance in the interactions between tomato and powdery mildew fungus. Upon O. neolycopersici infection, the intensive formation of H 2 O 2 was observed in the leaves of resistant tomato cultivars, but no or very low levels of H 2 O 2 molecules were produced in the leaves of susceptible cultivars [39,40]. Similarly, in this study, the accumulation of H 2 O 2 at the infection sites in the leaves of ShWRKY81-silenced plants was much lower than that in the control plants after On-Lz infection, implying that the expression levels of ShWRKY81 affect the generation of H 2 O 2 in the interactions between resistant tomato and On-Lz. The hypersensitive response (HR) is another defense response to block powdery mildew pathogen invasion via arresting fungal growth in the attacked host cells [41]. The silencing of a positive regulator of tomato resistance to On-Lz in LA1777 reduced the induction of HR and showed more white powdery spots than that in LA1777 seedlings [5,7,42]. Additionally, in this study, the silencing of ShWRKY81 resulted in a~30% decrease in the HR production at the infection sites after 48 hpi and led to a loss of resistance to On-Lz, supporting a positive role of ShWRKY81 in LA1777 immunity.
Although many WRKYs were demonstrated to function as important immune regulators in other species, most of them modulated plant resistance against various hemibiotrophic and necrotrophic pathogens. Conversely, this study elucidated the contribution of ShWRKY81 in wild tomato against biotrophic fungus (O. neolycopersici), enriching the roles of WRKYs in different species against multi-type pathogens. OsWRKY45 and AtWRKY70, the key components in rice and Arabidopsis immunity [19,43], are homologues of ShWRKY81 (Figure 2a), which is consistent with the function of ShWRKY81 in wild tomato against O. neolycopersici. ShWRKY81 responded in a different way to diverse abiotic stimuli, elevated transcripts in response to temperature changes, and down-regulated transcripts in response to NaCl or PEG6000 treatment, suggesting the role of ShWRKY81 in tomato tolerance to temperature, salinity, and drought stresses. Likewise, a genome-wide expression analysis revealed that the transcripts of SlWRKY81 were down-regulated in tomato (Solanum lycopersicum) under drought stress [44]. SlWRKY81 was reported to negatively regulate drought tolerance via attenuating proline accumulation and nitric oxide production in tomato [45,46]. It is not surprising that one transcription factor functions as a multifaceted player in different stresses. For example, OsWRKY76 negatively regulates innate immunity but confers chilling tolerance in rice [21,22,33]. OsWRKY76-overexpressing rice lines exhibited decreased resistance to M. oryzae and Xanthomonas oryzae pv oryzae, whereas it displayed enhanced tolerance to cold stress [21,22,33]. Although the transcripts of ShWRKY81 were up-regulated in LA1777 with exogenous JA treatment, the JA levels and the expression of JA signaling marker genes displayed no significant change in ShWRKY81-silenced plants upon On-Lz infection, which is consistent with previous findings that LA1777 does not rely on the JA signaling pathway to block powdery mildew infection [7], while SA levels were induced by On-Lz in LA1777, and the accumulation of this hormone was linked to the susceptibility to On-Lz in ShARPC3-silenced plants [5]. In addition, the transcripts of SA-inducible defense genes were triggered following On-Lz infection in LA1777 [5,7]. Based on these studies, SA levels and SA signaling are speculated to participate in tomato defense against O. neolycopersici.
SA is a critical phytohormone in defending against invading pathogens and plays a vital role in the activation of PTI, ETI, and systemic acquired resistance (SAR) [47]. The levels of SA are accumulated in plants upon pathogen infection, leading to the increased expression of downstream defense-related genes. In Arabidopsis, SA-deficient 2 (sid2) mutants showed abolished pathogen-induced SA accumulation and enhanced susceptibility to some virulent or avirulent bacteria and fungi [48,49]. Similarly, transgenic tobacco plants constitutively expressing the NahG gene, a salicylate hydroxylase, inhibited the accumulation of SA after inoculation and lost resistance to a large number of pathogens [50,51]. In addition, a recent study revealed that the overexpression of the SA-degrading enzyme hydroxylase 3 (OsSAH3) in rice blocked Magnaporthe oryzae-induced SA production and conferred increased susceptibility to several hemibiotrophic and necrotrophic pathogens [52]. In this study, the silencing of ShWRKY81 in wild tomato LA1777 led to a lower induction of SA accumulation and the expression of SA downstream pathogenesis-related genes, and more severe symptoms than in the control following On-Lz infection, suggesting that SA is essential for ShWRKY81-mediated resistance to On-Lz. Intriguingly, WRKY transcription factors were shown to serve as positive regulators of the SA biosynthesis gene, isochorismate synthase1 (ICS1). AtWRKY28 binds directly to the promoter of ICS1 based on chromatin immunoprecipitation (ChIP) assays and electrophoretic mobility shift assays (EMSA), and the overexpression of AtWRKY28 or AtWRKY46 in Arabidopsis protoplasts resulted in the enhanced expression of ICS1 [53]. However, whether or how ShWRKY81 directly regulates SA production in the defense response via the activation of SA biosynthesis genes need to be studied further.

Powdery Mildew Production for Tomato Infection
O. neolycopersici Lanzhou strain (On-Lz), isolated from tomato leaves with typical powdery mildew lesions in Gansu Province, China, was maintained and propagated on susceptible tomato leaves, as described by Sun et al. [5].

Production of Bacterial Strains Used for Gene Silencing
Escherichia coli strain DH5α carrying the pMD19T-ShWRKY81 vector was cultured at 37 • C for 12-16 h on Luria-Bertani (LB) media containing 50 µg/mL ampicillin. Agrobacterium tumefaciens strain GV3101 harboring a silencing-related construct was cultured at 28 • C for 48 h on yeast extract-peptone (YEP) media containing 50 µg/mL of rifampicin and kanamycin.

Plant Material and Growth Conditions
The On-Lz-resistant cultivar Solanum habrochaites cv. LA1777 and On-Lz susceptible cultivar Solanum lycopersicum cv. Money-maker (MM) were used in this research. After surface sterilization [54], tomato seeds were germinated in vermiculite for 7 days in a growth chamber at 22 • C under a 16 h light/8 h dark cycle with 80-90% relative humidity. Then, the tomato seedlings were transplanted to soil and grown in a glasshouse providing the same conditions mentioned above.

Inoculation and Treatment
For inoculation, fresh On-Lz spores were collected from infected tomato MM and used to prepare a suspension (5 × 10 4 conidia/mL) [55]. Immediately, the suspension was sprayed on the leaves of 4-week-old LA1777 and MM seedlings; then, these seedlings were grown in a glasshouse with the same conditions mentioned above. After inoculation, the samples were separately harvested at 0, 6,12,24,48,72,96, and 120 hpi (hours post inoculation). For hormone treatment, 4-week-old LA1777 seedlings were sprayed with 100 µM MeJA, 200 µM SA, and mock solution, respectively. The samples were separately harvested at 0, 0.5, 3, 6, 12, and 24 hpt (hours post treatment). For temperature treatment, 4-week-old LA1777 seedlings were moved to a growth chamber at 10 • C, 22 • C, or 40 • C under a 16 h light/8 h dark cycle with 80-90% relative humidity. For NaCl and PEG6000 treatment, the roots of 4-week-old LA1777 seedlings were poured with 200 mM NaCl, 20% (w/v) PEG6000, and mock solution, respectively. The samples were separately harvested at 0, 1, 3, 6, 12, and 24 hpt. All treatments mentioned above were repeated independently three times, and each replicate contained three seedlings.

Cloning of ShWRKY81 and Bioinformatic Analyses
The complete open-reading frame (ORF) of ShWRKY81 was amplified from LA1777 cDNA with the DNA primers (Supplemental Table S1) via PCR, and cloned to a pMD19T simple vector (Takara, Dalian, China), followed by DNA sequencing for validation. The primers were designed via the Primer Premier 6 software according to the SlWRKY81 mRNA sequence (NM_001279343.1) and synthesized in Tsingke Biotechnology company (Xi'an, China). The amino acid sequence of ShWRKY81 protein was submitted to online interfaces for bioinformatic analyses. The molecular weight and conserved domains were deduced using ProtParam (https://web.expasy.org/protparam/, accessed on 22 May 2018) and InterPro (http://www.ebi.ac.uk/interpro/, accessed on 22 May 2018), respectively. The signal peptide and subcellular localization were predicted by cNLS Mapper (http: //nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi, accessed on 22 May 2018) and Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/, accessed on 22 May 2018). The protein function predictions were obtained from PlantTFDB (http://planttfdb. gao-lab.org/, accessed on 14 January 2023). Homologs of ShWRKY81 were retrieved from GenBank databases using protein blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 14 January 2023). The multiple sequence alignment of ShWRKY81 and its homologues was carried out via DNAMAN6.0, and a phylogenetic dendrogram was generated with MEGA 7.0 using the neighbor-joining method.

Subcellular Localization Analysis
The coding region of ShWRKY81 was inserted into the p16318hGFP plasmid and fused with a green fluorescent protein (GFP) gene, generating an expression construct (35S::ShWRKY81-GFP). The obtained construct (35S::ShWRKY81-GFP) and control (35S::GFP) were transformed into A. tumefaciens strain GV3101 via the freeze-thaw method [56]. A. tumefaciens cultures harboring the plasmid mentioned above were separately mixed with equal A. tumefaciens cultures harboring 35S::NLS-dsRED in infiltration buffer (200 µM acetosyringone, 10 mM MES, and 10 mM MgCl 2 ) to a final OD600 of 0.5. After 3 h incubation at room temperature in darkness, the mixed cultures were infiltrated into four-week-old Nicotiana benthamiana leaves, respectively. Then, the N. benthamiana plants were kept in a growth chamber at 22 • C under a 16 h light/8 h dark cycle for 2 days. Green and red fluorescence were detected by a laser confocal scanning microscope (Olympus FV1000, Olympus, Tokyo, Japan).

Quantitative Real-Time PCR(qRT-PCR) Analysis
Total RNA was isolated from the samples mentioned above using Biozol RNA Reagent (Biomiga, Shanghai, China). Complementary DNA (cDNA) was generated from 2 µg of total RNA using a PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Dalian, China). qRT-PCR was performed using the Bio-Rad IQ™5 Real-Time PCR System (Bio-Rad, Hercules, CA, USA) with Ultra SYBR Mixture (CWBIO, Beijing, China). The qRT-PCR reaction mixture contained 2 µL template cDNA (200 ng/µL), 0.4 µL of each primer (10 µM), 10 µL 2 × Ultra SYBR Mixture, and 7.2 µL water in a final volume of 20 µL. The cDNA was amplified by 40 cycles of PCR (denaturation for 10 s at 95 • C, annealing for 30 s at 60 • C, and elongation for 30 s at 72 • C) following initial denaturation for 10 min at 95 • C. A non-template control was detected in each reaction, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference. All the experiments were carried out in biological triplicate and two technical replicates. The transcripts for each sample in qRT-PCR were normalized to the transcripts of ShGAPDH by the 2 −∆∆CT method [57]. The primers are listed in Supplemental Table S1.

TRV2-Mediated ShWRKY81 Silencing
A 322 bp fragment of ShWRKY81 was amplified from the plasmid pMD19T-ShWRKY81 with gene-specific primers (Supplemental Table S1), and then was inserted into the pTRV2 vector linearized via XbaI and XhoI to create the pTRV2::ShWRKY81 construct using the Seamless Cloning kit (Vazyme, Nanjing, China). The pTRV2::ShPDS construct was generated by the same method. The intended inserts of these constructs were examined by sequencing. All the gene silencing constructs were separately mobilized into A. tumefaciens strain GV3101 via the freeze-thaw method [56]. A. tumefaciens cultures harboring pTRV2 or pTRV2 constructs were equally mixed with A. tumefaciens cultures harboring pTRV1 in infiltration buffer (200 µM acetosyringone, 10 mM MES, and 10 mM MgCl 2 ) to a final OD 600 of 1.0. After 3 h incubation at room temperature in darkness, the mixed cultures were used to infiltrate the primary leaves of four-leaf-stage LA1777 seedlings [58,59]. pTRV2 and pTRV2::ShPDS constructs were used as controls. At~30 days after virus inoculation, the pTRV2::ShPDS control seedlings displayed photo-bleaching phenotypes, and the upper leaves of seedlings infiltrated with pTRV2 or pTRV2::ShWRKY81 were inoculated with On-Lz. The efficiency of gene silencing was examined in parallel by qRT-PCR using genespecific primers for ShWRKY81 (Supplemental Table S1). After inoculation, samples at 24, 48, and 120 hpi were harvested for expression analysis, and samples at 12 and 24 hpi were prepared for phytohormone extraction. The experiments were carried out three times, and each assay contained 6 seedlings with 5 inoculated leaves.

Histological and Phenotypic Observations
The responses of ShWRKY81-silenced LA1777 and control seedlings to On-Lz were observed. Following inoculation, samples at 24, 48, and 72 hpi were collected for histological observation. The 3,3-diaminobenzidine and trypan blue staining were separately carried out to assess the accumulation of H 2 O 2 and the induction of HR cell death, as previously described [60,61]. At least 50 infection sites from three leaves were examined at each time point using a Nikon Eclipse 80i microscope (Nikon Corporation, Tokyo, Japan). For phenotypic observations, disease severity was recorded with a disease rating scale (0-9) as follows: 0 = no indication of infection; 1 = leaves with a percentage of infected leaf area up to 0-5%; 3 = leaves with a percentage of infected leaf area up to 6-10%; 5 = leaves with a percentage of infected leaf area up to 11-20%; 7 = leaves with a percentage of infected leaf area up to 21-40%; and 9 = leaves with a percentage of infected leaf area up to 41-100% [62]. Disease index was statistically analyzed using the following equation: Disease index = [Σ (number of infected plant leaves at a scored disease severity × the disease severity)/(total number of counted plant leaves × 9)] × 100%. The average DI of each infected plant was analyzed at three independent time points.

Phytohormone Determination
The infected leaves of ShWRKY81-silenced LA1777 and control seedlings at 12 and 24 hpi were prepared for phytohormone analysis. The extraction and determination of JA and SA in the samples mentioned above were performed using the method as described by de Sá et al. [63]. The experiment was carried out independently three times.

Statistical Analyses
All statistical analyses in this research were performed via a Student's t-test (p < 0.05) using the SPSS ver. 20.0 software (IBM, New York, NY, USA).

Conclusions
We cloned an On-Lz-inducible WRKY transcription factor, ShWRKY81, and showed that the expression patterns of this gene responded to diverse abiotic stimuli and exogenous defense hormones in wild tomato LA1777. The knockdown of ShWRKY81 led to host susceptibility to On-Lz in LA1777, with a loss of H 2 O 2 formation, hypersensitive response