MdVQ12 confers resistance to Valsa mali by regulating MdHDA19 expression in apple

Abstract Valine‐glutamine (VQ) motif‐containing proteins play a crucial role in plant biotic stress responses. Apple Valsa canker, caused by the ascomycete Valsa mali, stands as one of the most severe diseases affecting apple trees. Nonetheless, the underlying resistance mechanism of VQ proteins against this disease has remained largely unexplored. This study reports MdVQ12, a VQ motif‐containing protein, as a positive regulator of apple Valsa canker resistance. Genetic transformation experiments demonstrated that MdVQ12 overexpression increased resistance to V. mali, while gene silencing lines exhibited significantly reduced resistance. MdVQ12 interacted with the transcription factor MdWRKY23, which bound to the promoter of the histone deacetylase gene MdHDA19, activating its expression. MdHDA19 enhanced apple resistance to V. mali by participating in the jasmonic acid (JA) and ethylene (ET) signalling pathways. Additionally, MdVQ12 promoted the transcriptional activity of MdWRKY23 towards MdHDA19. Our findings reveal that MdVQ12 enhances apple resistance to V. mali by regulating MdHDA19 expression and thereby regulating the JA and ET signalling pathways, offering potential candidate gene resources for breeding apple Valsa canker‐resistant germplasm.


| INTRODUC TI ON
In the plant immune system, the first line of defence against pathogenic microorganisms, pattern-triggered immunity (PTI) is mediated by membrane-localized pattern recognition receptors (PRRs) that recognize microbial-or pathogen-associated molecular patterns (MAMPs or PAMPs).Subsequently, effector-triggered immunity (ETI) recognizes pathogen effectors by intracellular nucleotidebinding leucine-rich repeat receptors (NLRs) to stimulate the immune response (Jones & Dangl, 2006).
When PTI or ETI is triggered, a cascade of signals will expand and extend from the site of invasion to the downstream of immunity, causing plants to limit pathogen colonization and invasion.
Transcription factors (TFs), especially WRKYs, play a critical role in this process.For example, in the immune response induced by flg22, there is a mitogen-activated protein kinases cascade (MAPK), an indispensable component in the process of plant immune signal transduction.The immune signal is transmitted downward through MEKK1, which affects the downstream TFs such as WRKY22 and WRKY29 to achieve immune signal transduction (Asai et al., 2002).
Phosphorylation of WRKY8 by MAPK functions in the defence response in Nicotiana benthamiana (Ishihama et al., 2011).These studies demonstrate that WRKY TFs play an important role in coordinating plant immunity signalling.
WRKY TFs are plant-specific factors that can control the transcription of various genes and participate in the regulation of a variety of plant life activities.They were first identified in sweet potato by Ishiguro and Nakamura (1994).Subsequently, several WRKY TFs have been reported in more than 20 plant species, including rice, Arabidopsis, and tomato (Huang et al., 2012;Wu et al., 2005).
WRKY TFs are important regulators of the defence response at the transcriptional level.Knockdown of CaWRKY1 in pepper resulted in a reduction of Xanthomonas growth in leaves, demonstrating its important regulatory role in pathogen-induced immune responses (Oh et al., 2010).WRKY11 and WRKY17 negatively regulate plant resistance to Pseudomonas syringae in Arabidopsis (Journot-Catalino et al., 2006).MdWRKY100 can improve leaf resistance to Colletotrichum gloeosporioides in apple (Zhang et al., 2019).However, the contribution and regulatory mechanism through which WRKY TFs are involved in protecting apple from V. mali invasion is not yet clear.
TFs often require some transcriptional regulators to assist their function, and VQ motif-containing proteins are one of the key proteins.VQ proteins play an important role in the response of plants to biotic stress (Jing & Lin, 2015).The transcription level of AtVQ23/SIB1 was strongly induced by P. syringae and Botrytis cinerea infection, and gene overexpression improved the disease resistance in Arabidopsis (Lai et al., 2011;Xie et al., 2010).
Overexpression of AtVQ10 enhanced resistance to B. cinerea, whereas the vq10 mutant reduced resistance to the pathogen (Chen et al., 2018).As transcriptional regulators, VQ proteins can interact with a variety of TFs, including WRKY, to participate in the response to biotic stress (Chi et al., 2013;Jing & Lin, 2015).For instance, AtVQ23 and AtVQ16 could interact and activate WRKY33 by enhancing its DNA-binding activity to improve plant disease resistance (Lai et al., 2011).Nevertheless, there are few studies on the joint regulatory roles of VQ proteins and WRKY TFs in resistance to V. mali.
Apple Valsa canker, caused by the fungus V. mali, is one of the most severe diseases of apple (Abe et al., 2007;Xu et al., 2020;Yin et al., 2016).In this study, MdVQ12 was upregulated during V. mali infection.Therefore, we thought that it might be involved in the regulation of resistance to apple Valsa canker.However, the regulatory mechanism of MdVQ12 to V. mali resistance remains a mystery.Here, we reported that MdVQ12 confers apple resistance to V. mali by regulating the expression of the histone deacetylase gene MdHDA19.
Histone acetylation plays an important role in plant epigenetic modification and this process is reversible, mainly including histone acetyltransferases (HATs) and histone deacetylases (HDACs) (Ma et al., 2013).Histone deacetylases are critical for plant growth, development, and stress response (Ma et al., 2013;Zhou et al., 2005).
This study provides new insights into the molecular mechanism of the resistance to V. mali and an important reference value for the breeding of apple disease resistance.

| MdVQ12 positively regulates apple resistance to V. mali
The analysis of MdVQ12's conserved domain identified a VQ domain (Figure S1a).Protein feature visualization showed that this protein is located inside the cytomembrane, indicating that it is an intracellular localization protein (Figure S1b).Subcellular localization analysis revealed that it was localized to the nucleus (Figure S1c).Furthermore, upregulation of MdVQ12 relative expression was detected during V. mali inoculation (Figure S1d), suggesting its involvement in the responses to V. mali infection.
Next, stable transgenic apple calli expressing MdVQ12 were established to characterize the function of MdVQ12 (Figure S2a).
Calli infected for 4 days were used to determine lesion area, H 2 O 2 , and O 2− levels.Results indicated that MdVQ12-OE-2/3/6 apple calli exhibited increased resistance to V. mali infection, with 31.4% ± 5.6%, 32.4% ± 6.3%, and 25.1% ± 6.4% reductions in lesion areas compared to the wild type (WT) (Figure S2b,c).Additionally, MdVQ12-overexpressing apple calli exhibited significantly higher levels of reactive oxygen species (ROS) compared with the WT (Figure S2d,e).We then made an association study by showing the R 2 and p values between levels of MdVQ12 transcripts and lesion area, H 2 O 2 content, and O 2− content (Table S1).These results demonstrated that MdVQ12's capacity to enhance resistance in apple calli against V. mali.
Stable transgenic GL-3 tissue culture seedlings were obtained for further investigating MdVQ12 functionality.Agarose gel electrophoresis experiments demonstrated the presence of DNA bands exclusively in MdVQ12-OE lines (Figure S3a).Western blot analysis confirmed the detection of MdVQ12-HA in MdVQ12overexpression (OE) lines (Figure S3b).Reverse transcriptionquantitative PCR (RT-qPCR) analysis revealed higher expression of MdVQ12 in MdVQ12-OE lines compared to the WT (Figure S3c).
The previous results confirm that MdVQ12 positively regulates apple resistance against V. mali.

MdWRKY23-dependent
VQ proteins modulate plant disease resistance by interacting with TFs to regulate their transcriptional activities (Jing & Lin, 2015;Li et al., 2014).Therefore, to explore the molecular mechanism of MdVQ12 in V. mali resistance, we employed immunoprecipitationmass spectrometry (IP-MS) to identify interacting TFs.Among the candidate proteins, the WRKY TF MdWRKY23 demonstrated interaction with MdVQ12 in yeast two-hybrid (Y2H) assays (Figure 2a).This interaction was further confirmed through coimmunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays.Co-IP assay results showed MdVQ12-HA detection only in the presence of MdWRKY23-GFP (Figure 2b).
Additionally, the BiFC assay revealed fluorescence when MdVQ12 and MdWRKY23 were co-expressed, confirming their interaction (Figure 2c).These results establish that MdVQ12 interacts with  S2).This underscores the essential role of the WRKY domain in facilitating the MdWRKY23-MdVQ12 interaction.
To investigate the relationship between MdVQ12 and MdWRKY23 in V. mali resistance, MdWRKY23 was silenced while overexpressing MdVQ12 in GL-3 tissue culture seedling leaves.Interestingly, the enhanced resistance to V. mali conferred by MdVQ12 alone was lost after MdWRKY23 silencing (Figure S5), indicating that MdVQ12's ability to enhance apple resistance is contingent upon the presence of MdWRKY23.

| MdWRKY23 can bind to the MdHDA19 promoter to activate its expression
DNA affinity purification sequencing (DAP-seq) identified genes bound by MdWRKY23, elucidating its role in regulating resistance against V. mali.MdHDA19 (a histone deacetylase gene) emerged as a downstream target, and was confirmed via electrophoretic mobility shift assay (EMSA) and yeast one-hybrid (Y1H) assays (Figure 3a,b).

| MdHDA19 positively modulates apple resistance to V. mali
Histone acetylation is regulated by histone acetyltransferases and deacetylases, which crucially regulates gene expression.HDA19 has been reported to positively modulate the resistance to the fungal pathogen Alternaria in Arabidopsis by regulating several ethylene (ET) and jasmonic acid (JA) signal transduction-related genes to participate in the ET and JA signalling pathways (Zhou et al., 2005).
Therefore, we hypothesized that MdHDA19 similarly contributes to apple Valsa canker resistance.To explore the function of MdHDA19, we conducted transient expression assays.The transgenic leaves of GL-3 tissue culture seedlings of MdHDA19 and empty vectors (EVs) were generated (Figure S6a,c) and infected with V. mali for 32 h.Results confirmed that MdHDA19-OE-1/5 apple leaves exhibited enhanced resistance to V. mali, with lesion areas decreasing by 42.2% ± 3.9% and 54.4% ± 2.2% compared to EV.Conversely, lesion areas in MdHDA19-RNAi-2/5 apple leaves were 64.4% ± 14.1% and 55% ± 11.7% larger than EV (Figure S6b,d).In addition, we made an association study by showing the R 2 and p values between levels of MdHDA19 transcripts and lesion area (Table S1).These results demonstrated that MdHDA19 bolsters GL-3 leaf resistance against V. mali.
To explore whether MdHDA19 is involved in ET and JA signalling pathways, we measured the relative expression of ET ( MdERF1) and JA (MdCOI1, MdMYC2, MdLOX3, and MdVSP2 transduction-related genes in apple leaves.The results showed that they were all upregulated when MdHDA19 was overexpressed compared with the EVs (Figure S7).Because MdHDA19 is a histone deacetylase, we investigated whether it has histone deacetylase activity and whether the activation of this activity is related to the ET and JA pathways.In apple leaves overexpressing MdHDA19, we quantitatively measured the expression levels of genes related to the ET and JA pathways after treatment with the histone deacetylase inhibitor trichostatin A (TSA).The results demonstrated that after TSA treatment, the expression levels of related genes were significantly decreased in the apple leaves overexpressing MdHDA19, with some even decreasing to levels close to the control (Figure S8).These findings indicate that MdHDA19 possesses histone deacetylase activity and its activation is associated with both ET and JA signalling pathways.
These results demonstrate that MdHDA19 can enhance the resistance of apple calli to V. mali.Similarly, we measured the expression levels of ET and JA signal transduction-related genes in apple calli.Consistent with the results of GL-3 leaves, they were also upregulated in the MdHDA19-overexpressing apple calli compared with the WT (Figure 5); therefore, we thought that MdHDA19 was involved in the ET and JA signalling pathways and thus enhanced apple resistance to V. mali.

| MdVQ12 functions as a positive transcriptional regulator of MdWRKY23 and confers apple resistance to V. mali by activating the ET and JA signalling pathways
Because VQ proteins usually act in conjunction with TFs to affect the transcriptional activity of TFs (Jing & Lin, 2015;Lei et al., 2017) control approach.VQ proteins regulate many aspects of plant growth and development, including plant disease resistance, by interacting with TFs, modulating their transcriptional activity (Chi et al., 2013;Jing & Lin, 2015;Lai et al., 2011;Li et al., 2014).
However, the molecular mechanisms of VQ proteins in V. mali resistance remain unclear.In this study, we found that MdVQ12 confers apple resistance to V. mali by facilitating MdWRKY23's transcriptional activation of MdHDA19 in the ET and JA signalling pathways.
Previous studies have shown that the function of VQ proteins is largely affected by the VQ motif (FxxhVQxhTG) (Jing & Lin, 2015).There is a conserved VQ motif in MdVQ12; therefore, MdVQ12 should putatively have the typical functions of VQ proteins.VQ proteins regulate plant immunity, for example, AtVQ21 overexpression in Arabidopsis enhanced resistance to P. syringae (Andreasson et al., 2005) but reduced resistance to B. cinerea (Fiil & Petersen, 2011;Petersen et al., 2010).In our study, MdVQ12 increased apple resistance to V. mali, contributing to VQ protein research in plant immunity.VQ proteins function by recruiting other TFs (i.e., MdWRKY23) that activate downstream genes involved in disease resistance (Lai et al., 2011;current study).Here, we found that MdVQ12 could interact with MdWRKY23.
The WRKY domain in WRKY TFs is required for protein-protein interactions (Eulgem et al., 2000).Here, we established that the WRKY domain in MdWRKY23 is essential for the MdWRKY23-MdVQ12 interaction.The cis-acting element W-box ([C/T] TGAC [C/T]) in gene promoters serves as a binding site for WRKY TFs, and mutations within TGAC can impair binding (Rushton et al., 2010).Our DAP-seq results revealed the presence of a Wbox in the promoter of the histone deacetylase gene MdHDA19, and MdWRKY23 was found to bind to it, thereby activating MdHDA19 expression.Previous studies have demonstrated that HDACs exhibit histone deacetylase activity attributed to their role as histone deacetylases, and this activity is closely associated with ET and JA signalling.For instance, Arabidopsis HDA6 displays histone deacetylase activity, which can globally influence histone acetylation levels.HDA6 mutant and RNAi plants exhibit higher levels of histone acetylation compared to the WT, and the expression of ET and JA signalling-related genes is downregulated (Wu et al., 2008).Similarly, Arabidopsis HDA19 also possesses histone deacetylase activity.Overexpression of HDA19 leads to reduced histone acetylation levels compared to the WT, enhanced resistance to the fungal pathogen Alternaria, and upregulation of ET and JA signalling-related genes.Conversely, HDA19 RNAi plants show increased histone acetylation levels and downregulated expression of ET and JA signalling-related genes (Zhou et al., 2005).Therefore, we propose that MdHDA19 has histone deacetylase activity and its activation is associated with both ET and JA signalling pathways.
In this study, we found that several ET and JA signalling-related genes were upregulated in MdHDA19-overexpressing apple leaves and calli, leading to increased resistance to the fungal pathogen V. mali.Additionally, these genes exhibited significantly decreased expression levels after TSA treatment.These results indicate that MdHDA19 has histone deacetylase activity and that the activation of this activity is related to the ET and JA signalling pathways, ultimately enhancing apple resistance to the pathogenic fungus V. mali.Moreover, consistent with previous reports that VQ proteins can modulate the transcriptional activity of TFs, we demonstrated that MdVQ12 could promote the transcriptional activation of MdWRKY23 on MdHDA19.However, there are no reports on the relationship between VQ proteins and HDACs.Therefore, this study offers novel insights into the regulatory mechanism of VQ proteins.
In plant defence, ROS burst and callose deposition are fundamental processes crucial for the defensive reaction (Boller & Felix, 2009;Schwessinger & Ronald, 2012).Callose deposition increases cell wall thickness, thereby slowing pathogen invasion, making it a universal model for quantifying plant defence responses (Luna et al., 2011;Nishimura et al., 2003).ROS play a central role in defending against pathogen invasion and activating plant immune responses, conferring resistance (Chen et al., 1993;Kariola et al., 2005;Qi et al., 2017;Sharma et al., 2012).VQ proteins, functioning as transcriptional regulators, typically co-regulate plant immune responses along with their interacting TFs, including ROS burst and callose deposition.In this study, we confirmed that MdVQ12 was induced during V. mali

| Plant and microbe materials
The apple tissue culture seedlings are of the GL-3 genotype (Dai et al., 2013).The GL-3 plantlets and apple cv.Orin calli were cultured on Murashige and Skoog (MS) medium at 25°C.N. benthamiana seedlings were grown in a growth chamber and the V. mali WT strain 03-8 (Yin et al., 2015) were grown in incubators at 25°C.
MdWRKY23.F I G U R E 1 MdVQ12 positively regulates apple resistance to Valsa mali.(a) Disease symptoms and lesion areas/lengths of wild type (WT), MdVQ12-OE, and MdVQ12-RNAi apple leaves and twigs at 36 h post-inoculation (hpi) and 48 hpi, respectively.(b) H 2 O 2 contents of WT, MdVQ12-OE, and MdVQ12-RNAi apple leaves at 36 hpi.(c) O 2− contents and production rates of WT, MdVQ12-OE, and MdVQ12-RNAi apple leaves at 36 hpi.(d) Callose contents of WT, MdVQ12-OE, and MdVQ12-RNAi apple leaves at 36 hpi.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; t test.Data are shown as mean ± SD.In the study, MdWRKY23 was divided into seven fragments, and the VQ domain was removed from MdVQ12 to assess the necessity of WRKY and VQ domains for the interaction between MdWRKY23 and MdVQ12 via Y2H assays.Results indicate that the WRKY domain combining the N-terminal segment can interact with MdVQ12 (Table MdVQ12 can interact with MdWRKY23.(a) MdVQ12 can interact with MdWRKY23 in yeast two-hybrid (Y2H) assays.(b) MdVQ12 can interact with MdWRKY23 in co-immunoprecipitation (Co-IP) assays.(c) MdVQ12 can interact with MdWRKY23 in bimolecular fluorescence complementation (BiFC) assays.Bars = 20 μm.
, we investigated MdVQ12's influence on the transcriptional activity of MdWRKY23 by detecting luminescence signals and LUC activity.N. benthamiana leaves were co-infiltrated with various combinations: combination 1, combination 2, and combination 2 together with MdVQ12-pGreenII 62-SK (combination 5).The results showed that both luminescence signal and LUC activity were stronger when F I G U R E 3 MdWRKY23 can bind to the MdHDA19 promoter to activate its expression.(a) MdWRKY23 can bind to the MdHDA19 promoter in electrophoretic mobility shift (EMSA) assays.(b) MdWRKY23 can bind to the MdHDA19 promoter in yeast onehybrid (Y1H) assays.(c) MdWRKY23 can activate MdHDA19 expression in luciferase (LUC) imaging and LUC activity assays.(d) MdWRKY23 can activate MdHDA19 expression in β-glucuronidase (GUS) staining and GUS activity assays.***p < 0.001; t test.Data are shown as mean ± SD.F I G U R E 4 MdHDA19 confers apple calli resistance to Valsa mali.(a) Relative expression of MdHDA19.(b) Disease symptoms of wild-type (WT) and MdHDA19-OE apple calli at 3 days post-inoculation (dpi).(c) Lesion areas of WT and MdHDA19-OE apple calli at 3 dpi.(d) H 2 O 2 contents of WT and MdHDA19-OE apple calli at 3 dpi.(e) O 2− contents of WT and MdHDA19-OE apple calli at 3 dpi.Bars with different letters are significantly different at p < 0.05 according to one-way analysis of variance (Tukey's test).Data are shown as mean ± SD.F I G U R E 5 Relative expression of genes related to the jasmonic acid (JA) and ethylene (ET) signalling pathways.Bars with different letters are significantly different at p < 0.05 according to one-way analysis of variance (Tukey's test).Data are shown as mean ± SD. combination 5 was co-expressed than when combination 2 was coexpressed (Figure 6a,b), indicating that MdVQ12 functions as a positive transcriptional regulator of MdWRKY23.As MdWRKY23 was identified to transcriptionally activate MdHDA19, and MdVQ12 acts as a positive transcriptional regulator of MdWRKY23, we assessed the relative expression levels of MdHDA19 in MdVQ12-overexpressing lines.The results indicated a significant upregulation of MdHDA19 (Figure 6c).We postulated that MdVQ12 may also modulate the ET and JA signalling pathways mediated by MdHDA19.Accordingly, we quantitatively assessed genes associated with the ET and JA signalling pathways.The results showed a significant increase in their accumulation in MdVQ12-overexpressing lines (Figure 6d), suggesting that MdVQ12 enhances apple resistance to V. mali by regulating MdHDA19 expression and thereby activating the ET and JA signalling pathways.Taken together, MdVQ12 was able to activate MdHDA19mediated ET and JA signalling pathways by enhancing the transcriptional activation activity of MdWRKY23 on MdHDA19, which in turn further enhanced the resistance against V. mali. 3 | DISCUSS ION Apple Valsa canker, attributed to V. mali, inflicts significant economic losses.The poor efficiency and effectiveness of traditional control methods have limited apple production.Identifying disease resistance genes remains the most cost-effective disease F I G U R E 6 MdVQ12 functions as a positive transcriptional regulator of MdWRKY23.(a) Luciferase (LUC) imaging assay detecting the transcriptional activity of MdWRKY23.(b) LUC activity assay detecting the transcriptional activity of MdWRKY23.(c) Relative expression of MdHDA19 in wild-type (WT) and MdVQ12-OE apple leaves.(d) Relative expression of genes related to the jasmonic acid (JA) and ethylene (ET) signalling pathways in WT and MdVQ12-OE apple leaves.(e) Working model showing MdVQ12 confers apple resistance to Valsa mali by modulating the expression of MdHDA19.Bars with different letters are significantly different at p < 0.05 according to one-way analysis of variance (Tukey's test).**p < 0.01, ***p < 0.001; t test.Data are shown as mean ± SD.
infection and significantly enhanced ROS and callose accumulation, advancing our understanding of VQ protein-mediated disease resistance.The results of our regulatory approach are summarized in a model in Figure 6e.MdVQ12 interacts with MdWRKY23 to form a complex in this model.This interaction modulates the transcriptional capacity of MdWRKY23 towards MdHDA19, and they are novel components of the regulatory network of apple Valsa canker resistance.Furthermore, MdHDA19 contributes to apple resistance against V. mali by participating in the JA and ET signalling pathways.In conclusion, our study establishes that MdVQ12 acts as an activator within the MdWRKY23-MdHDA19 module, which mediates apple Valsa canker resistance.This work provides a novel regulatory network for understanding disease modulation by VQ proteins.Our findings offer theoretical guidance and technical support for the cultivation of disease-resistant germplasm resources.