Transient Silence of VvCSN5 Enhances Powdery Mildew Resistance in Grapevine (Vitis Vinifera)

Transient Silence of VvCSN5 Enhances Powdery Mildew Resistance in Grapevine (Vitis Vinifera) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Transient Silence of VvCSN5 Enhances Powdery Mildew Resistance in Grapevine ( Vitis Vinifera ) Kai-Cheng Cui, Min Liu, Gui-Hua Ke, Xing-Yuan Zhang, Bo Mu, Min Zhou, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-284658/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract As one of the most economically important fruit crops in the world, grapevine ( Vitis vinifera ) suffers significant yield losses from many pathogens including powdery mildew caused by Erysiphe necator . By contrast, several wild Chinese grapevines including Vitis pseudoreticulata accession ‘Baihe-35-1’ exhibit a high resistance to powdery mildew pathogen. Here, we identified a grapevine gene CSN5 ( COP9 signalosome complex subunit 5 ), designated VvCSN5 , which showed different expression patterns in ‘Baihe-35-1’ and in susceptible cultivar V. vinifera ‘Thompson Seedless’ during powdery mildew isolate En NAFU1 infection. Moreover, transient silence of VvCSN5 in ‘Thompson Seedless’ leaves enhanced resistance to En NAFU1, which is accompanied by cell wall deposition at the attempt sites, and hypersensitive response-like cell death of penetrated epidermal cells. Several defense-related marker genes ( VvPR1 , VvPR3 , VvPAD4 , and VvRBOHD ) had higher basal expression levels in VvCSN5 -silenced leaves. In addition, we found the structure and activity of CSN5 promoters in ‘Thompson Seedless’ and ‘Baihe-35-1’ were discrepant, which may be one of the reasons for their different resistance to powdery mildew infection. Taken together, these results imply that grapevine CSN5 plays an important role in the responses to powdery mildew. Plant Physiology and Morphology Plant Molecular Biology and Genetics Grapevine Powdery mildew (Erysiphe necator) CSN5 Transient transformation Promoter Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Grapevine is one of the most important fruit crops in the world, with over 7000 years cultivation history (Jaillon et al. 2007 ). Among all grapevine species, Eurasian grapevine species Vitis vinifera is the main cultivar for viticulture and wine production on account of its superior aroma and flavor characteristics. However, V. vinifera is vulnerable to various diseases, including powdery mildew caused by an obligate biotrophic fungus Erysiphe necator , which infects all green tissues of grapevine, causes a widespread and devastating disease (Gadoury et al. 2012 ; Qiu et al. 2015 ). In contrast, several wild Chinese grapevines species have a strong powdery mildew resistance, such as Vitis pseudoreticulata accession Baihe-35-1 (Gao et al. 2016 ; Hu et al. 2019 ), which is therefore an important germplasm for enhancing powdery mildew resistance of V. vinifera through genetic improvement. To date, several powdery mildew resistance gene loci, including RUN1 , RUN2 , REN5 in Muscadinia rotundifolia (Barker et al. 2005 ; Riaz et al. 2011 ; Blanc et al. 2012 ), and REN4 , REN6 , REN7 in V. romanetii and V. piasezkii (Ramming et al. 2011 ; Pap et al. 2016 ; Mahanil et al. 2012 ) had been identified through forward genetic approaches. However, the molecular mechanisms research of grapevine resistance (R) genes is difficult because of its characteristic of perennial woody plants with a heterozygous genome (Feechan et al. 2013 ). Plants have an innate immune system to avoid pathogen infection, and the two major branches of which are PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI) (Jones and Dangl 2006 ). The first branch of PTI uses transmembrane pattern recognition receptors (PRRs) to recognize multiple conserved microbial- or pathogen-associated molecular patterns (MAMPs and PAMPs) and cause penetration resistance eventually (Zipfel and Felix 2005 ). The second branch of ETI relies on a large amount of nucleotide-binding leucine-rich-repeat (NB-LRR) proteins encoded by R-genes, which can identify the effectors released from a pathogen and then lead to hypersensitive response (HR) (Jones and Dangl 2006 ; Dangl and Jones 2001 ). As described above, the powdery mildew resistance in grapevine is also composed of penetration resistance and hypersensitive response (Douchkov et al. 2016 ; Wang et al. 2016 ). These two processes are interacted and impede the growth of powdery mildew by cell wall deposition and programmed cell death (Jones and Dangl 2006 ; Boutrot and Zipfel 2017 ; Hu et al. 2019 ). COP9 signalosome complex subunit 5 (CSN5) is one of the eight subunits of COP9 (constitutive photomorphogenesis 9) signalosome (CSN) in Arabidopsis . It participated in regulating CULLIN-RING E3 ubiquitin ligases (CRLs) activity as the catalytic center of the complex (Wei and Deng 2003 ; Cope et al. 2002 ; Gusmaroli et al. 2007 ). As the most conservative subunit in CSN, CSN5 has become a common target of pathogenic effectors (Echalier et al. 2013 ; Jin et al. 2014 ). In Arabidopsis , AtCSN5a interacts with 29 distinct effectors from Hyaloperonospora arabidopsidis ( Hpa ) and Pseudomonas syringae ( Psy ), and AtCSN5a mutations increase resistance to both Hpa and Psy (Mukhtar et al. 2011 ). The role of CSN5 in biotic stress responses has also been reported in tobacco (Liu et al. 2002 ), tomato (Shang et al. 2019 ), wheat (Zhang et al. 2017 ), and rice (He et al. 2020 ). However, there is no report focus on CSN5 regulating defense response in grapevine. In the present study, we identified a grapevine gene CSN5 which showed different expression patterns in susceptible and resistant grapevine during powdery mildew infection. Through transient transformation, we discovered that silencing VvCSN5 enhanced powdery mildew resistance in susceptible cultivar V. vinifera ‘Thomspon Seedless’ leaves. Further analysis of the promoter structure and function indicated that the differences in CSN5 promoter between ‘Thomspon Seedless’ and ‘Baihe-35-1’ may be one of the reasons for their different resistance to powdery mildew. Our findings suggested that grapevine CSN5 negatively regulated powdery mildew resistance. Materials And Methods Plant materials and growth conditions The tissue culture seedlings of V. vinifera cv. Thompson Seedless and V. pseudoreticulata accession Baihe-35-1 were transplanted in individual pots containing soil mix (peat: perlite: vermiculite, 4:1:1, v:v:v), and grown in a illumination incubator for two weeks with temperatures ranging from 22°C to 26°C, under a 14 h/10 h (light/dark) light cycle. Cloning of grapevine CSN5 genes The grapevine CSN5 genes from ‘Thompson Seedless’ ( VvCSN5 ) and ‘Baihe-35-1’ ( VpCSN5 ) were amplified from corresponding leaves cDNA by Planta Max Super-Fidelity DNA Polymerase (Vazyme Bio Co., Nanjing, China), respectively. The amplified primers were derived from the CSN5 gene sequence we uploaded to the National Center for Biotechnology Information (NCBI) database ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ). The PCR products were cloned into the pMD19-T vector (Takara Bio Inc., Dalian, China) and sequenced at the Beijing AuGCT Biotech, Yangling, Sequencing Department, then aligned with the genome of V. vinifera PN40024 ( https://www.ncbi.nlm.nih.gov/genome ). The sequence of amplified primers and related GeneBank accession number were shown in Table S1 and Table S2, respectively. Plasmid construction To obtain over-expression recombinant vector, full length VpCSN5 gene was amplified from the pMD19-T-VpCSN5 fusion plasmid, and inserted into Bam HI and Kpn I sites in the pCAMBIA2300 vector containing the CaMV 35S promoter and green fluorescent protein (GFP) coding sequence by homologous recombination to generate 35S::VpCSN5-GFP. To silence VvCSN5 in ‘Thompson Seedless’, the vector pK7WIWG2D was used as the RNA interference vector. A 358 bp length fragment from pMD19-T-VvCSN5 was selected and amplified for constructing pK7WIWG2D -VvCSN5 vector through Invitrogen Gateway recombination cloning technology (Thermo Fisher Inc., Carlsbad, USA). To analyze the promoters of VvCSN5 and VpCSN5 , we designed the primers according to the genome of V. vinifera PN40024, and amplified about 1500 bp promoter sequences upstream the initiation codon of CSN5 gene from ‘Thompson Seedless’ and ‘Baihe-35-1’, respectively. The PCR products were then cloned into the pMD19-T vector and sequenced. To construct the reporter plasmids for GUS activity analysis, we cloned the VvCSN5 promoter (ProTS) and VpCSN5 promoter (ProBH) sequences into pBI121 vector to drive encoding β-glucuronidase (GUS) gene, generated construct ProTS:GUS and ProBH:GUS . The pBI121 vector was pre-digested by Hind III and Bam HI enzyme. The sequence of related primers was shown in Table S1. Subcellular location assay To explore the subcellular localization of grapevine CSN5, we used the 35S::VpCSN5-GFP plasmid that mentioned above, and transiently expressed in grapevine mesophyll protoplasts was performed as previous study (Zhao et al. 2016 ; Liu et al. 2019 ), the 35S:GFP as a control. The transformed protoplasts were incubated for 20–25 h at room temperature in the dark, then observed by Olympus BX51 fluorescence microscopy (Japan). Agrobacterium -mediated transient transformation and inoculation Agrobacterium tumefaciens strain GV3101 was used for the transient transformation of related plasmids. Firstly, centrifuged the shaken bacterial solution (OD600, 0.5–0.6) and resuspensed it with an equal volume of induction buffer (3mM Na 2 HPO 4 , 50mM MES (pH 5.6), 0.5% (w/v) Glucose, 100µM Acetosyringone), 28°C, 180 rpm shaking for one hour. Then, chose the third or fourth fully expanded leaves, carefully injected the suspending liquid into back of leaves with a disposable sterile syringe. The method of transient transformation referred to previous studies (Kurth et al. 2012 ; Krenek et al. 2015 ; Bertazzon et al. 2012 ; Urso et al. 2013 ; Xie et al. 2020 ). Three days later, transformed leaves were inoculated with fresh conidia of powdery mildew isolate En NAFU1, which through touching the epidermis of the transformed leaves using heavily infected ‘Thompson Seedless’ leaves (Gao et al. 2016 ). Evaluation of resistance to powdery mildew Three groups of transformed leaves, pK7WIWG2D-VvCSN5 (RNAi), 35S::VpCSN5-GFP (OE) and 35S::GFP (as a control, EV) were assessed for resistance to En NAFU1 (Gao et al. 2016 ). 3,3’-diaminobenzidine (DAB) and trypan blue (TB) stained the epidermal cells of transformed leaves were observed at 2 and 5 days post inoculation (dpi) to identify the accumulation of H 2 O 2 , fungal structures and dead host cells. Aniline blue-staining was used to visualize the accumulation of callose at 5 dpi (Gao et al. 2016 ; Hu et al. 2018 ; Wan et al. 2020 ). All the samples were then examined using an Olympus BX51 microscope (Japan). Hyphal length of En NAFU1 and frequencies of the invasion induced hypersensitive cell death (full of H 2 O 2 ) were directly measured under Olympus BX51 using the CellSens operation software (Hu et al. 2018 ). Quantitative real-time PCR (RT-qPCR) analysis To explore the expression patterns of VvCSN5 and VpCSN5 during the powdery mildew infection, we selected disease-free leaves from ‘Thompson Seedless’ and ‘Baihe-35-1’ for powdery mildew isolate En NAFU1 inoculation. Then collected leaves with 0, 12, 24, 48, 72, 96, 120 hours post inoculation (hpi) in turn, samples were promptly stored in -80℃ refrigerator after quick freezing by liquid nitrogen. The sampling method of RT-qPCR for defense-related genes was the same as above. Total RNA was extracted from collected leaves using E.Z.N.A. Plant RNA Kit (Omega, Guangzhou, China). The cDNA was obtained by reverse transcription using HiScript Q Select RT SuperMix (Vazyme, Nanjing, China). RT-qRCR assays were refer to previous study (Zhang et al. 2015 ). Grapevine ACTIN7 (GenBank accession number is XM_002282480.4) was used as an internal control (Wang et al. 2017 ; Gutha et al. 2010 ; Reid et al. 2006 ). The relative transcript levels of the genes were calculated using the 2 −△△Ct and Normalized Expression method. Each sample was analyzed in three biological replications. The statistical significance was evaluated by Student’s t -test. The sequence of RT-qPCR primers and gene accession number were shown in Table S1 and S2, respectively. Histochemical and fluorometric assays for GUS activity The histochemical GUS assay of leaves was carried out as previously described (Jefferson 1987 ). The VvCSN5 promoter: GUS ( ProTS:GUS ) and VpCSN5 promoter: GUS ( ProBH:GUS ) vector were transformed into fully unfolded healthy ‘Thomspon Seedless’ or tobacco leaves by vacuumizing and 35S:GUS was used as a control. Three days after transformation, the transformed leaves were inoculated s by spraying the En NAFU1 spore suspension with a concentration of 5 × 10 5 sporangia ml − 1 . Mock-inoculated leaves were sprayed with sterile water only. Collected 2 dpi leaves, placed in GUS dye and incubated at 37℃ for 24 hours. Then, 70% and 100% ethanol were successively used for decoloring at 37℃ for 10 hours. The method of GUS quantitative analysis was referred to previous study (Yu et al. 2013 ). Statistical analysis The relevant data were analyzed through Student’s t -test or Tukey’s HSD test using the statistics software tool IBM SPSS v26.0.0. The mean values ± standard deviation of the mean (SD) was calculated based on the results of at least three independent experiments, and significant differences compared with controls are represented by p < 0.05 and p < 0.01. Results Bioinformatics analysis and subcellular localization of grapevine CSN5 Although there are two CSN5 homologous genes in Arabidopsis thaliana ( AtCSN5a and AtCSN5b ), only one CSN5 gene in V. vinifera genome (Jin et al. 2014 ), we named VvCSN5 . It's located on chromosome 3, the full length of genomic DNA is 8398 bp with six exons and five introns (Fig. 1 a). To understand the association of CSN5 in various plants, we firstly chosen nine homologous CSN5 proteins from seven species which functions had been well studied, and used them to build a phylogenetic tree with VvCSN5 from ‘Thompson Seedless’ and VpCSN5 from ‘Baihe-35-1’. We found VvCSN5 and VpCSN5 were highly homologous with their homologous proteins (Fig. 1 b). VvCSN5, VpCSN5 and other CSN5 proteins were also highly conserved at their functional region (MPN domain) (Fig. 1 c). These results indicated that grapevine CSN5 may play similar roles to those homologous genes. It was reported that CSN5 is localized in cytoplasm and nucleus in most plants (Li et al. 2018 ; Wang et al. 2013 ). In order to explore the intracellular localization of grapevine CSN5, we transiently expressed VpCSN5-GFP fusion protein in ‘Baihe-35-1’ grapevine mesophyll protoplasts. As shown in Fig. 1 d, the GFP expressed from the control construct was dispersed throughout the whole cell, while VpCSN5-GFP fusion protein localized to both cytoplasm and nucleus. The expression patterns of VpCSN5 and VvCSN5 are different during powdery mildew infection To determine whether there is any difference in expression patterns of VvCSN5 and VpCSN5 after powdery mildew infection, we inoculated the susceptible grapevine ‘Thompson Seedless’ and resistant grapevine ‘Baihe-35-1’ using isolate En NAFU1, respectively. RT-qPCR results showed that the VvCSN5 transcript lightly increased in abundance and reached a peak of ~ 1.4-fold at 24 hpi, while VpCSN5 transcript decreased rapidly at 24 hpi (0.4-fold) and then returned to a normal level at 48 hpi (Fig. 2 ). This suggested that grapevine CSN5 may be involved in the defense responses to powdery mildew. Transient silence of VvCSN5 enhances resistance to powdery mildew isolate En NAFU1 in grapevine As above mentioned, we have known that the expression patterns of CSN5 in susceptible and resistant grapevines were discrepant (Fig. 2 ). Whereupon, we wondered whether altering the expression levels of CSN5 in ‘Thompson Seedless’ leaves could affect their resistance to powdery mildew. We thus obtained VvCSN5-RNAi (RNAi) and 35S::VpCSN5-GFP (OE) transient transformed leaves through injection, construct 35S::GFP (EV) was injected as control (Fig. 3 a). As shown in Fig. 3 b, most epidermal cells of injection range of ‘Thompson Seedless’ leaf for three constructs injected showed clear GFP signals. Similarly, the western blot analysis of GFP (∼29.9 kDa) or VpCSN5-GFP fusion protein (∼70.2 kDa) also confirmed the normal expression of related GFP protein (Fig. 3 c), which means that the transient transformation was successful. Subsequently, we detected the transcript levels of CSN5 in each transformed leaf or uninjected leaf (as control) by RT-qPCR. The results showed that CSN5 transcript levels were up-regulated over 20-fold in OE leaves and down-regulated to 0.5-fold in RNAi leaves compared with control (Fig. 3 d). To assess the resistance to powdery mildew in leaves with different CSN5 levels,we collected transformed ‘Thompson Seedless’ leaves stained with trypan blue (TB) and 3,3’-diaminobenzidine (DAB), and measured the total hyphal length from isolated fungal colonies. As shown in Fig. 4 a and b, the total hyphal length per colony in the leaves of RNAi was ∼600 µm at 2 dpi, while the length in the leaves of OE and EV were ∼950 and 840 µm, respectively. Moreover, at 5 dpi, nearly 20% of invaded cells showed whole-cell H 2 O 2 -staining in RNAi leaves, while both the leaves of EV and OE were less than 5% (Fig. 4 a and c). In addition, cell wall deposition by callose accumulation is also an important indicator of Vitis resistance to powdery mildew (Consonni et al. 2006 ). The aniline blue-staining results showed that around the necrotic epidermal cells, major callose accumulated in RNAi leaves at 5 dpi. In contrast, there was no obviously callose accumulated in the leaves of EV and OE (Fig. 4 d). These results suggested that silencing VvCSN5 could enhance the resistance to powdery mildew in grapevine. Grapevine CSN5 involve in multiple defenses signaling pathways To investigate which defense-related genes were affected by CSN5, we selected several typical defense-related genes to analyze their expression patterns in transformed leaves of ‘Thompson Seedless’. As shown in Fig. 5 , for SA-associated genes, VvPR1 (pathogenesis-related gene 1) showed a higher expression levels (over 2-fold) in RNAi leaves than that in EV at 0 dpi and remained high levels at 2 and 5 dpi, while the VvPR1 level in OE was lower (~ 0.5-fold) than EV in each time point. VvPR3 (pathogenesis-related gene 3) and VvPAD4 (phytoalexin deficient 4) transcript levels in RNAi leaves was both slightly higher (~ 1.5-fold) than that in EV at 0 dpi, except that VvPR3 levels at 5 dpi was not obviously differ from EV. However, the JA-associated gene VvJAR1 (jasmonate resistant 1) showed a stronger expression (1.3 ~ 2.5-fold) in OE leaves than that in EV and RNAi. For hydrogen peroxide-associated gene VvRBOHD (respiratory burst oxidase homologue D), the increasing expression trend between RNAi and EV was similar, but the transcript levels at 0, 2, 5 dpi in RNAi was all significantly higher (1.5 ~ 2-fold) than that in EV, which implied hydrogen peroxide-pathway had a stronger activation in RNAi leaves. In addition, the ethylene-associated gene VvACS2 (1-amino-cyclopropane-1-carboxylate synthase 2) transcript level in RNAi was lightly higher than EV at 2 dpi. These results indicated that grapevine CSN5 was associated with multiple defense-related genes during powdery mildew infection. The promoters of VvCSN5 and VpCSN5 are different in structure and activity According to the sequence alignment, we found there are only two different amino acid residues between VvCSN5 and VpCSN5 (Fig. S1). Thus, we wonder whether the different expression patterns between VvCSN5 and VpCSN5 due to the differences of their promoters. We firstly compared the promoters of VvCSN5 (ProTS) and VpCSN5 (ProBH) by sequence alignment. As shown in Fig. 6 a, there were masses of differences between these two promoter sequences, including a number of base pair substitutions and a 132 bp fragment deletion in the VpCSN5 promoter. To further analyze these two sequences, we forecasted the cis-regulatory elements (CREs) of the two promoters by PlantCare ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) and markered out the discrepant elements (Fig. 6 a). Some of them, such as MYB-, MYC- and ABI3-elements, had been reported to be responsive to various stresses (Dubos et al. 2010 ; Abe et al. 2003 ; Tamminen et al. 2001 ).. To verified the function of promoters, we used histochemical staining and fluorometric assays to examine expression of the GUS genes which respectively driven by ProTS or ProBH after inoculation with powdery mildew isolate En NAFU1. As shown in Fig. 6 b and c, the 35S:GUS control leaves were strongly stained by GUS dye, and the quantitative GUS assay showed similar activities in En NAFU1-inoculated and mock conditions. Furthermore, the ProTS:GUS in mock condition showed a very weak expression, while in En NAFU1-inoculated leaves showed a much strong activity which was 3.5-fold higher than in mock (Fig. 6 b and c). However, there was no significant difference in ProBH:GUS activity between mock and En NAFU1-inoculated leaves (Fig. 6 b and c). We also expressed ProTS:GUS and ProBH:GUS in tobacco leaves (for a higher transformation efficiency) with mock condition to confirm the reliability of GUS staining results of grapevine leaves. We discovered stronger staining in tobacco leaves than that in corresponding grapevine leaves (Fig. 6 b), and similar to mock in grapevine leaves, the ProBH:GUS activity in tobacco was also higher than ProTS:GUS (Fig. 6 c). These results implied that the differences in expression patterns of CSN5 between susceptible and resistant grapevine may cause by different promoter activation. Discussion As the catalytic center of COP9 signalosome (CSN), CSN5 plays an essential role for normal working of complex (Stratmann and Gusmaroli 2012 ; Jin et al. 2014 ). Thus, CSN5 is quite conserved in eukaryotes (Cope et al. 2002 ). We already know that CSN5 is involved in the process of pathogen defense in many plants, including Arabidopsis (Mukhtar et al. 2011 ), tobacco (Liu et al. 2002 ), wheat (Zhang et al. 2017 ), rice (He et al. 2020 ), etc. However, there was no related research on grapevine CSN5. In this study, we demonstrated the conservation of CSN5, and further discovered that it was associated with grapevine resistance to powdery mildew. According to the expression analysis of grapevine CSN5 during powdery mildew infection, we found that the VpCSN5 expression reached a minimum at 24 hpi, whereas VvCSN5 reached a maximum at 24 hpi (Fig. 2 ), which was consistent with the timing of haustorial formation (Hu et al. 2019 ). This result implies that the changes of CSN5 expression are pathogen induced. Further combining with the results of stronger powdery mildew resistance of CSN5-silenced leaves (Fig. 3 ), which both supporting a negative role of CSN5 on powdery mildew infection. Previous studies showed CSN5 was mainly involved in plant pathogen-defense through JA- or SA-signaling pathways (Feng et al. 2003 ; Hind et al. 2011 ; Spoel et al. 2009 ). Interestingly, the effects of CSN5 to the two pathways are opposite. For example, the over-expression of OsCSN5a inhibits rice black-streaked dwarf virus (RBSDV) infection through activating JA-signaling pathway in rice (He et al. 2020 ). Whereas silencing TaCSN5 in wheat improves wheat leaf rust resistance by activation of SA-signaling pathway (Zhang et al. 2017 ). In our study, a stronger powdery mildew resistance showed in CSN5-RNAi leaves and three SA-associated marker genes ( VvPR1 , VvPR3 , VvPAD4 ) had higher basal expression levels (Figs. 4 and 5 ), which suggested that the role of grapevine CSN5 in pathogen defense is probably similar to TaCSN5 in wheat. In addition, the JA-associated marker gene VvJAR1 showed stronger expression in CSN5 over-expression leaves (Fig. 5 ), which suggested grapevine CSN5 may be positively correlated with JA-signaling pathway, like its homologous genes in Arabidopsis (Feng et al. 2003 ). Interestingly, there is only one different amino acid residue in the MPN domain of VvCSN5 and VpCSN5 (Fig. S1), however, there were significant differences in their expression patterns during powdery mildew infection (Fig. 2 ). We know that gene expression depends on its promoter, in which pathogen-inducible gene expression requires the interaction between defense-related transcription factors (TFs) and cis-regulatory elements (CREs) in the promoter (Amorim et al. 2017 ; Rushton et al. 2002 ). For example, promoter mutations in rice Xa13 gene cause down-regulation of expression during host–pathogen interaction, resulting in the race-specific resistance to Xanthomonas oryzae pv. Oryzae (Chu et al. 2006 ). In grapevine, ‘Baihe-35-1’ VpRFP1 gene expressed under its own promoter activated a stronger disease resistance than driven by V. vinifera cv. Carignane VvRFP1 promoter (Yu et al. 2013 ). In this study, we found that there were many base pair substitutions and a 132 bp fragment deletion in VpCSN5 promoter that resulted in more CAAT-box and stress-related CREs existed in VvCSN5 promoter sequence than that in VpCSN5 promoter (Fig. 6 a), including MYB-elements and ERE-elements, which were reported to be responsive to salicylic acid and ethylene (Yang and Klessig 1996 ; Fujimoto et al. 2000 ), respectively. This explained the reason of VvCSN5 promoter displayed a strong inducible GUS activity in response to powdery mildew (Fig. 6 b and c). Our study expounded the effect of CSN5 on grapevine resistance to powdery mildew, confirmed the functional consistence between grapevine CSN5 and its homologous genes. Further demonstrate the different structures of CSN5 promoters between susceptible and resistant grapevines caused CSN5 gene differently expressed during powdery mildew infection. Taken together, our results suggested grapevine CSN5 was a negative regulator gene for grapevine powdery mildew resistance. Declarations Author contribution statement CKC and YQW conceived and designed the research. CKC and ML conducted most of the experiments. GHK, XYZ, BM and YH participated in the experiments. CKC wrote the manuscript. All authors read and approved the manuscript. Acknowledgements This study was funded by the National Natural Science Foundation of China (Grant No. 31972986 to YQW). Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. References Abe H, Urao T, Ito T, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell 15(1):63–78. doi: 10.1105/tpc.006130 Amorim LLB, dos Santos RD, Neto JPB, Guida-Santos M, Crovella S, Benko-Iseppon AM (2017) Transcription Factors Involved in Plant Resistance to Pathogens. Curr Protein Pept Sc 18(4):335–351. doi: 10.2174/1389203717666160619185308 Barker CL, Donald T, Pauquet J, Ratnaparkhe MB, Bouquet A, Adam-Blondon AF, Thomas MR, Dry I (2005) Genetic and physical mapping of the grapevine powdery mildew resistance gene, Run1, using a bacterial artificial chromosome library. TAG Theoretical and applied genetics Theoretische und angewandte Genetik 111 (2):370–377. doi: 10.1007/s00122-005-2030-8 Bertazzon N, Raiola A, Castiglioni C, Gardiman M, Angelini E, Borgo M, Ferrari S (2012) Transient silencing of the grapevine gene VvPGIP1 by agroinfiltration with a construct for RNA interference. Plant Cell Rep 31(1):133–143. doi: 10.1007/s00299-011-1147-2 Blanc S, Wiedemann-Merdinoglu S, Dumas V, Mestre P, Merdinoglu D (2012) A reference genetic map of Muscadinia rotundifolia and identification of Ren5, a new major locus for resistance to grapevine powdery mildew. TAG Theoretical and applied genetics Theoretische und angewandte Genetik 125 (8):1663–1675. doi: 10.1007/s00122-012-1942-3 Boutrot F, Zipfel C (2017) Function, Discovery, and Exploitation of Plant Pattern Recognition Receptors for Broad-Spectrum Disease Resistance. Annu Rev Phytopathol 55:257–286. doi: 10.1146/annurev-phyto-080614-120106 Chu Z, Yuan M, Yao J, Ge X, Yuan B, Xu C, Li X, Fu B, Li Z, Bennetzen JL, Zhang Q, Wang S (2006) Promoter mutations of an essential gene for pollen development result in disease resistance in rice. Genes Dev 20(10):1250–1255. doi: 10.1101/gad.1416306 Consonni C, Humphry ME, Hartmann HA, Livaja M, Durner J, Westphal L, Vogel J, Lipka V, Kemmerling B, Schulze-Lefert P, Somerville SC, Panstruga R (2006) Conserved requirement for a plant host cell protein in powdery mildew pathogenesis. Nat Genet 38(6):716–720. doi: 10.1038/ng1806 Cope GA, Suh GS, Aravind L, Schwarz SE, Zipursky SL, Koonin EV, Deshaies RJ (2002) Role of predicted metalloprotease motif of Jab1/Csn5 in cleavage of Nedd8 from Cul1. Science 298(5593):608–611. doi: 10.1126/science.1075901 Dangl JL, Jones JD (2001) Plant pathogens and integrated defence responses to infection. Nature 411(6839):826–833. doi: 10.1038/35081161 Douchkov D, Lueck S, Hensel G, Kumlehn J, Rajaraman J, Johrde A, Doblin MS, Beahan CT, Kopischke M, Fuchs R, Lipka V, Niks RE, Bulone V, Chowdhury J, Little A, Burton RA, Bacic A, Fincher GB, Schweizer P (2016) The barley (Hordeum vulgare) cellulose synthase-like D2 gene (HvCslD2) mediates penetration resistance to host-adapted and nonhost isolates of the powdery mildew fungus. New Phytol 212(2):421–433. doi: 10.1111/nph.14065 Dubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, Lepiniec L (2010) MYB transcription factors in Arabidopsis. Trends Plant Sci 15(10):573–581. doi: 10.1016/j.tplants.2010.06.005 Echalier A, Pan Y, Birol M, Tavernier N, Pintard L, Hoh F, Ebel C, Galophe N, Claret FX, Dumas C (2013) Insights into the regulation of the human COP9 signalosome catalytic subunit, CSN5/Jab1. Proc Natl Acad Sci U S A 110(4):1273–1278. doi: 10.1073/pnas.1209345110 Feechan A, Anderson C, Torregrosa L, Jermakow A, Mestre P, Wiedemann-Merdinoglu S, Merdinoglu D, Walker AR, Cadle-Davidson L, Reisch B, Aubourg S, Bentahar N, Shrestha B, Bouquet A, Adam-Blondon AF, Thomas MR, Dry IB (2013) Genetic dissection of a TIR-NB-LRR locus from the wild North American grapevine species Muscadinia rotundifolia identifies paralogous genes conferring resistance to major fungal and oomycete pathogens in cultivated grapevine. Plant J 76(4):661–674. doi: 10.1111/tpj.12327 Feng S, Ma L, Wang X, Xie D, Dinesh-Kumar SP, Wei N, Deng XW (2003) The COP9 signalosome interacts physically with SCF COI1 and modulates jasmonate responses. Plant Cell 15(5):1083–1094. doi: 10.1105/tpc.010207 Fujimoto SY, Ohta M, Usui A, Shinshi H, Ohme-Takagi M (2000) Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC box-mediated gene expression. Plant Cell 12(3):393–404. doi:Doi 10.2307/3870944 Gadoury DM, Cadle-Davidson L, Wilcox WF, Dry IB, Seem RC, Milgroom MG (2012) Grapevine powdery mildew (Erysiphe necator): a fascinating system for the study of the biology, ecology and epidemiology of an obligate biotroph. Mol Plant Pathol 13(1):1–16. doi: 10.1111/j.1364-3703.2011.00728.x Gao YR, Han YT, Zhao FL, Li YJ, Cheng Y, Ding Q, Wang YJ, Wen YQ (2016) Identification and utilization of a new Erysiphe necator isolate NAFU1 to quickly evaluate powdery mildew resistance in wild Chinese grapevine species using detached leaves. Plant physiology biochemistry: PPB 98:12–24. doi: 10.1016/j.plaphy.2015.11.003 Gusmaroli G, Figueroa P, Serino G, Deng XW (2007) Role of the MPN subunits in COP9 signalosome assembly and activity, and their regulatory interaction with Arabidopsis cullin3-based E3 ligases. Plant Cell 19(2):564–581. doi: 10.1105/tpc.106.047571 Gutha LR, Casassa LF, Harbertson JF, Naidu RA (2010) Modulation of flavonoid biosynthetic pathway genes and anthocyanins due to virus infection in grapevine (Vitis vinifera L.) leaves. Bmc Plant Biology 10. doi:Artn 187 1186/1471-2229-10-187 He L, Chen X, Yang J, Zhang T, Li J, Zhang S, Zhong K, Zhang H, Chen J, Yang J (2020) Rice black-streaked dwarf virus-encoded P5-1 regulates the ubiquitination activity of SCF E3 ligases and inhibits jasmonate signaling to benefit its infection in rice. New Phytol 225(2):896–912. doi: 10.1111/nph.16066 Hind SR, Pulliam SE, Veronese P, Shantharaj D, Nazir A, Jacobs NS, Stratmann JW (2011) The COP9 signalosome controls jasmonic acid synthesis and plant responses to herbivory and pathogens. Plant J 65(3):480–491. doi: 10.1111/j.1365-313X.2010.04437.x Hu Y, Gao YR, Yang LS, Wang W, Wang YJ, Wen YQ (2019) The cytological basis of powdery mildew resistance in wild Chinese Vitis species. Plant Physiol Biochem 144:244–253. doi: 10.1016/j.plaphy.2019.09.049 Hu Y, Li Y, Hou F, Wan D, Cheng Y, Han Y, Gao Y, Liu J, Guo Y, Xiao S, Wang Y, Wen YQ (2018) Ectopic expression of Arabidopsis broad-spectrum resistance gene RPW8.2 improves the resistance to powdery mildew in grapevine (Vitis vinifera). Plant Sci 267:20–31. doi: 10.1016/j.plantsci.2017.11.005 Jaillon O, Aury JM, Noel B, Policriti A, Clepet C, Casagrande A, Choisne N, Aubourg S, Vitulo N, Jubin C, Vezzi A, Legeai F, Hugueney P, Dasilva C, Horner D, Mica E, Jublot D, Poulain J, Bruyere C, Billault A, Segurens B, Gouyvenoux M, Ugarte E, Cattonaro F, Anthouard V, Vico V, Del Fabbro C, Alaux M, Di Gaspero G, Dumas V, Felice N, Paillard S, Juman I, Moroldo M, Scalabrin S, Canaguier A, Le Clainche I, Malacrida G, Durand E, Pesole G, Laucou V, Chatelet P, Merdinoglu D, Delledonne M, Pezzotti M, Lecharny A, Scarpelli C, Artiguenave F, Pe ME, Valle G, Morgante M, Caboche M, Adam-Blondon AF, Weissenbach J, Quetier F, Wincker P, French-Italian Public Consortium for Grapevine Genome C (2007) The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature 449 (7161):463–467. doi: 10.1038/nature06148 Jefferson R (1987) Assaying chimeric genes in plants: the GUS Gene Fusion System. Plant Mol Biol Rep 5(4):387–405 Jin D, Li B, Deng XW, Wei N (2014) Plant COP9 signalosome subunit 5, CSN5. Plant Sci 224:54–61. doi: 10.1016/j.plantsci.2014.04.001 Jones JD, Dangl JL (2006) The plant immune system. Nature 444(7117):323–329. doi: 10.1038/nature05286 Krenek P, Samajova O, Luptovciak I, Doskocilova A, Komis G, Samaj J (2015) Transient plant transformation mediated by Agrobacterium tumefaciens: Principles, methods and applications. Biotechnol Adv 33(6 Pt 2):1024–1042. doi: 10.1016/j.biotechadv.2015.03.012 Kurth EG, Peremyslov VV, Prokhnevsky AI, Kasschau KD, Miller M, Carrington JC, Dolja VV (2012) Virus-derived gene expression and RNA interference vector for grapevine. J Virol 86(11):6002–6009. doi: 10.1128/JVI.00436-12 Li Y, Chu ZN, Luo JY, Zhou YH, Cai YJ, Lu YG, Xia JH, Kuang HH, Ye ZB, Ouyang B (2018) The C2H2 zinc-finger protein SlZF3 regulates AsA synthesis and salt tolerance by interacting with CSN5B. Plant Biotechnol J 16(6):1201–1213. doi: 10.1111/pbi.12863 Liu J, Zhao FL, Guo Y, Fan XC, Wang YJ, Wen YQJPC, Tissue, Culture O (2019) The ABA receptor-like gene VyPYL9 from drought-resistance wild grapevine confers drought tolerance and ABA hypersensitivity in Arabidopsis. 138 (3):543–558 Liu Y, Schiff M, Serino G, Deng XW, Dinesh-Kumar SP (2002) Role of SCF ubiquitin-ligase and the COP9 signalosome in the N gene-mediated resistance response to Tobacco mosaic virus. Plant Cell 14(7):1483–1496. doi: 10.1105/tpc.002493 Mahanil S, Ramming D, Cadle-Davidson M, Owens C, Garris A, Myles S, Cadle-Davidson L (2012) Development of marker sets useful in the early selection of Ren4 powdery mildew resistance and seedlessness for table and raisin grape breeding. TAG Theoretical and applied genetics Theoretische und angewandte Genetik 124 (1):23–33. doi: 10.1007/s00122-011-1684-7 Mukhtar MS, Carvunis AR, Dreze M, Epple P, Steinbrenner J, Moore J, Tasan M, Galli M, Hao T, Nishimura MT, Pevzner SJ, Donovan SE, Ghamsari L, Santhanam B, Romero V, Poulin MM, Gebreab F, Gutierrez BJ, Tam S, Monachello D, Boxem M, Harbort CJ, McDonald N, Gai L, Chen H, He Y, European U, Effectoromics C, Vandenhaute J, Roth FP, Hill DE, Ecker JR, Vidal M, Beynon J, Braun P, Dangl JL (2011) Independently evolved virulence effectors converge onto hubs in a plant immune system network. Science 333(6042):596–601. doi: 10.1126/science.1203659 Pap D, Riaz S, Dry IB, Jermakow A, Tenscher AC, Cantu D, Olah R, Walker MA (2016) Identification of two novel powdery mildew resistance loci, Ren6 and Ren7, from the wild Chinese grape species Vitis piasezkii. BMC Plant Biol 16(1):170. doi: 10.1186/s12870-016-0855-8 Qiu W, Feechan A, Dry I (2015) Current understanding of grapevine defense mechanisms against the biotrophic fungus (Erysiphe necator), the causal agent of powdery mildew disease. Hortic Res 2:15020. doi: 10.1038/hortres.2015.20 Ramming DW, Gabler F, Smilanick J, Cadle-Davidson M, Barba P, Mahanil S, Cadle-Davidson L (2011) A single dominant locus, ren4, confers rapid non-race-specific resistance to grapevine powdery mildew. Phytopathology 101(4):502–508. doi: 10.1094/PHYTO-09-10-0237 Reid KE, Olsson N, Schlosser J, Peng F, Lund ST (2006) An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development. Bmc Plant Biology 6. doi:Artn 2710.1186/1471-2229-6-27 Riaz S, Tenscher AC, Ramming DW, Walker MA (2011) Using a limited mapping strategy to identify major QTLs for resistance to grapevine powdery mildew (Erysiphe necator) and their use in marker-assisted breeding. TAG Theoretical and applied genetics Theoretische und angewandte Genetik 122 (6):1059–1073. doi: 10.1007/s00122-010-1511-6 Rushton PJ, Reinstadler A, Lipka V, Lippok B, Somssich IE (2002) Synthetic plant promoters containing defined regulatory elements provide novel insights into pathogen- and wound-induced signaling. Plant Cell 14(4):749–762. doi: 10.1105/tpc.010412 Shang Y, Wang K, Sun S, Zhou J, Yu JQ (2019) COP9 Signalosome CSN4 and CSN5 Subunits Are Involved in Jasmonate-Dependent Defense Against Root-Knot Nematode in Tomato. Front Plant Sci 10:1223. doi: 10.3389/fpls.2019.01223 Spoel SH, Mou Z, Tada Y, Spivey NW, Genschik P, Dong X (2009) Proteasome-mediated turnover of the transcription coactivator NPR1 plays dual roles in regulating plant immunity. Cell 137(5):860–872. doi: 10.1016/j.cell.2009.03.038 Stratmann JW, Gusmaroli G (2012) Many jobs for one good cop - the COP9 signalosome guards development and defense. Plant Sci 185–186:50–64. doi: 10.1016/j.plantsci.2011.10.004 Tamminen I, Makela P, Heino P, Palva ET (2001) Ectopic expression of ABI3 gene enhances freezing tolerance in response to abscisic acid and low temperature in Arabidopsis thaliana. Plant J 25(1):1–8. doi:DOI 10.1046/j.1365-313x.2001.00927.x Urso S, Zottini M, Ruberti C, Lo Schiavo F, Stanca AM, Cattivelli L, Valè G (2013) An Agrobacterium tumefaciens-mediated gene silencing system for functional analysis in grapevine. Plant Cell Tissue Organ Cult 114(1):49–60. doi: 10.1007/s11240-013-0305-3 Wan DY, Guo Y, Cheng Y, Hu Y, Xiao S, Wang Y, Wen YQ (2020) CRISPR/Cas9-mediated mutagenesis of VvMLO3 results in enhanced resistance to powdery mildew in grapevine (Vitis vinifera). Hortic Res 7:116. doi: 10.1038/s41438-020-0339-8 Wang J, Yao W, Wang L, Ma F, Tong W, Wang C, Bao R, Jiang C, Yang Y, Zhang J, Xu Y, Wang X, Zhang C, Wang Y (2017) Overexpression of VpEIFP1, a novel F-box/Kelch-repeat protein from wild Chinese Vitis pseudoreticulata, confers higher tolerance to powdery mildew by inducing thioredoxin z proteolysis. Plant Sci 263:142–155. doi: 10.1016/j.plantsci.2017.07.004 Wang J, Yu Y, Zhang Z, Quan R, Zhang H, Ma L, Deng XW, Huang R (2013) Arabidopsis CSN5B interacts with VTC1 and modulates ascorbic acid synthesis. Plant Cell 25(2):625–636. doi: 10.1105/tpc.112.106880 Wang T, Chang C, Gu C, Tang S, Xie Q, Shen QH (2016) An E3 Ligase Affects the NLR Receptor Stability and Immunity to Powdery Mildew. Plant Physiol 172(4):2504–2515. doi: 10.1104/pp.16.01520 Wei N, Deng XW (2003) The COP9 signalosome. Annu Rev Cell Dev Biol 19:261–286. doi: 10.1146/annurev.cellbio.19.111301.112449 Xie YG, Ma YY, Bi PP, Wei W, Liu J, Hu Y, Gou YJ, Zhu D, Wen YQ, Feng JY (2020) Transcription factor FvTCP9 promotes strawberry fruit ripening by regulating the biosynthesis of abscisic acid and anthocyanins. Plant Physiol Biochem 146:374–383. doi: 10.1016/j.plaphy.2019.11.004 Yang Y, Klessig DF (1996) Isolation and characterization of a tobacco mosaic virus-inducible myb oncogene homolog from tobacco. Proc Natl Acad Sci U S A 93(25):14972–14977. doi: 10.1073/pnas.93.25.14972 Yu Y, Xu W, Wang J, Wang L, Yao W, Xu Y, Ding J, Wang Y (2013) A core functional region of the RFP1 promoter from Chinese wild grapevine is activated by powdery mildew pathogen and heat stress. Planta 237(1):293–303. doi: 10.1007/s00425-012-1769-9 Zhang H, Wang X, Giroux MJ, Huang L (2017) A wheat COP9 subunit 5-like gene is negatively involved in host response to leaf rust. Mol Plant Pathol 18(1):125–133. doi: 10.1111/mpp.12467 Zhang K, Han YT, Zhao FL, Hu Y, Gao YR, Ma YF, Zheng Y, Wang YJ, Wen YQ (2015) Genome-wide Identification and Expression Analysis of the CDPK Gene Family in Grape, Vitis spp. BMC Plant Biol 15:164. doi: 10.1186/s12870-015-0552-z Zhao FL, Li YJ, Hu Y, Gao YR, Zang XW, Ding Q, Wang YJ, Wen YQ (2016) A highly efficient grapevine mesophyll protoplast system for transient gene expression and the study of disease resistance proteins. Plant Cell Tiss Org 125(1):43–57. doi: 10.1007/s11240-015-0928-7 Zipfel C, Felix G (2005) Plants and animals: a different taste for microbes? Curr Opin Plant Biol 8(4):353–360. doi: 10.1016/j.pbi.2005.05.004 Supplementary Files SupplementaryinformationFigureS1.docx SupplementaryinformationTableS1.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 03 Mar, 2021 Reviewers invited by journal 02 Mar, 2021 Editor assigned by journal 01 Mar, 2021 First submitted to journal 27 Feb, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-284658","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":14886733,"identity":"7937ebb5-5c3f-4c18-9472-2cfb16c14159","order_by":0,"name":"Kai-Cheng Cui","email":"","orcid":"","institution":"Northwest Agriculture and Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kai-Cheng","middleName":"","lastName":"Cui","suffix":""},{"id":14886734,"identity":"5d0c5455-02c7-4fd2-874b-66aae2df3046","order_by":1,"name":"Min Liu","email":"","orcid":"","institution":"Northwest Agriculture and Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Liu","suffix":""},{"id":14886735,"identity":"d2917564-98ab-4dbd-bf68-719f0271e650","order_by":2,"name":"Gui-Hua Ke","email":"","orcid":"","institution":"Northwest Agriculture and Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gui-Hua","middleName":"","lastName":"Ke","suffix":""},{"id":14886736,"identity":"3721990d-7ed3-4623-a27e-e24aa88e40b7","order_by":3,"name":"Xing-Yuan Zhang","email":"","orcid":"","institution":"Northwest Agriculture and Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xing-Yuan","middleName":"","lastName":"Zhang","suffix":""},{"id":14886737,"identity":"3f880512-08e8-4322-9e2b-b2524da1ccd7","order_by":4,"name":"Bo Mu","email":"","orcid":"","institution":"Northwest Agriculture and Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Mu","suffix":""},{"id":14886738,"identity":"9a098623-5539-4517-8d37-a3a59340d03b","order_by":5,"name":"Min Zhou","email":"","orcid":"","institution":"Northwest Agriculture and Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Zhou","suffix":""},{"id":14886739,"identity":"eff21b95-b07e-4609-98e1-e3b9d868a079","order_by":6,"name":"Yang Hu","email":"","orcid":"","institution":"Northwest Agriculture and Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Hu","suffix":""},{"id":14886740,"identity":"3eff6e46-9421-420c-9aa9-8fa78508ba7a","order_by":7,"name":"Ying-Qiang Wen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYNCCCgj1gYGBmVgtZ8Ak4wzitTC2kaLF4HjvMYm38+4kzp+R/LCBocI6sYH97AH8Ws6cS5Ocu+1Z4oYbaYYNDGfSExt48hLwajG7kWMmzbvtcO4GiQTzB4xthxMbJHgM8Gu5/waoZc7h3Pkz0j82MP4jRssNHqCWhsO5DTdyDBsYG4jQYn8mx9hyzrHD9RvOvClsSDiWbtzGk4Nfi2T7GcMbb2oOG8u3p29s+FBjLdvPfga/FiBgkeCBMROAmI2QeiBg/sBDWNEoGAWjYBSMZAAAQb9LOYDoesIAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-7503-7260","institution":"Northwest Agriculture and Forestry University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Ying-Qiang","middleName":"","lastName":"Wen","suffix":""}],"badges":[],"createdAt":"2021-02-28 02:33:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-284658/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-284658/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":6850021,"identity":"fb2b70a1-38d5-4b7a-b824-74791b26fb4f","added_by":"auto","created_at":"2021-03-11 17:06:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":426831,"visible":true,"origin":"","legend":"Sequence analysis and localization of grapevine CSN5. (a) Location of VvCSN5 on chromosomes and its basic structure. (b) Phylogenetic analysis to ‘Thomspon Seedless’ VvCSN5, ‘Baihe-35-1’ VpCSN5 and several well-studied homologous proteins. The phylogenetic tree was constructed using the neighbor-joining method with 1000 bootstrap replicates by MEGA-X. (c) Alignment of the conserved core region (MPN domain) for the CSN5 proteins. Fully or partially conserved amino acid residues are shaded in black or gray, respectively. The MPN domain is marked by black underline. The multiple sequence alignment was made by ClustalX. (d) Subcellular localization of VpCSN5. The VpCSN5 gene were cloned from ‘Baihe-35-1’ and used to construct 35S::VpCSN5-GFP vector in which GFP was fused at the C terminus. The VpCSN5 fused proteins and GFP control were transiently expressed in grape mesophyll protoplasts and observed by fluorescence microscopy. Individual and merged images of GFP (first panel column) and chlorophyll auto-fluorescence (second column) as well as bright field images (column on the right) of protoplasts are shown (bars = 10 μm). The GenBank accession numbers of the genes mentioned in figure are presented at the Table S2. ","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-284658/v1/0fc4703faea886d7f078c662.png"},{"id":6849413,"identity":"09632225-68d0-4b29-9086-0b8e5b8737ec","added_by":"auto","created_at":"2021-03-11 17:03:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":43175,"visible":true,"origin":"","legend":"The different expression patterns of CSN5 gene in susceptible cultivar ‘Thompson Seedless’ and resistance grapevine ‘Baihe-35-1’ after En NAFU1 inoculation. Relative mRNA expression levels were measured by RT-qPCR. Grapevine ACTIN7 gene was used as an endogenous control. Each data point represents the mean ± standard deviation of triple biological replicates. Asterisks indicate that the transcript at each timepoint was significantly up- or down-regulated in comparison to 0 hpi (** and * = P≤0.01 and 0.05, respectively; Student's t-test). ","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-284658/v1/56e94df6d297ae2e61e0cdec.png"},{"id":6850019,"identity":"3945768e-260d-4bf8-8b0a-965df1044060","added_by":"auto","created_at":"2021-03-11 17:06:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":371828,"visible":true,"origin":"","legend":"Detection of transient transformation efficiency of ‘Thomspon Seedless’ leaves. (a) Comparison of the leaves 3 days after injection. From left to right is 35S::GFP (EV), pK7WIWG2D-CSN5 (RNAi) and 35S::CSN5-GFP (OE), respectively. (b) GFP fluorescence detection of transformed leaves (bars = 50 μm). (c) Western blot detection for protein expression levels of GFP (∼29.9kDa) or CSN5-GFP fusion protein (∼70.2kDa) in different transformed leaves. (d) The mRNA transcript levels of grapevine CSN5 in each transformed leaf. WT represents non-injected ‘Thomspon Seedless’ leaves under the same growth conditions. Asterisks indicate that the transcript of CSN5 at each group was significantly up- or down-regulated in comparison to WT (** = P≤0.01; Student's t-test). Samples of (b)(c)(d) were taken from the leaves 3 days after injection. \n\n","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-284658/v1/13dbcf399a968a9b98f34589.png"},{"id":6849418,"identity":"9680e272-ac19-40e5-ab55-db40f71a5215","added_by":"auto","created_at":"2021-03-11 17:03:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":441266,"visible":true,"origin":"","legend":"Transient silence of CSN5 improved ‘Thompson Seedless’ resistance to En NAFU1. (a) Representative micrographs showing DAB- and trypan blue-stained epidermal cells of the RNAi, OE and EV leaves at 2 or 5 dpi. Red arrowheads indicate H2O2 accumulation (bars = 50 μm). (b) Average hyphal length per colony of En NAFU1 inoculated on different groups leaves at 2 dpi. (c) Frequencies of the invasion induced hypersensitive cell death (full of H2O2) at 5 dpi. Data are means ± SE, calculated from three duplicated experiments. Asterisks indicate values significantly difference from EV (** and * = P≤0.01 and 0.05, respectively; Student's t-test). (d) Aniline blue-staining visualized the accumulation of callose in transformed leaves inoculated with En NAFU1 at 5 dpi. The images are respectively bright field, GFP, callose fluorescence and row 2 and row 3 merge image from the top to bottom (bars = 50 μm). ","description":"","filename":"Slide4.png","url":"https://assets-eu.researchsquare.com/files/rs-284658/v1/597f15629fea032f8b245f88.png"},{"id":6850024,"identity":"969f38fb-6e11-461a-932f-3794171b4bbc","added_by":"auto","created_at":"2021-03-11 17:06:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":89429,"visible":true,"origin":"","legend":"The expression levels of several defense-associated genes in RNAi, OE and EV leaves post En NAFU1 inoculation. Each data point represents the mean ± standard deviation of triple biological replicates. Grapevine ACTIN7 was used as an endogenous control. The GenBank accession numbers of the genes mentioned in figure are presented at the Table S2. Asterisks indicate that the mRNA expression of corresponding gene at each timepoint was significantly up- or down-regulated in comparison to EV (** and * = P≤0.01 and 0.05, respectively; Student's t-test). ","description":"","filename":"Slide5.png","url":"https://assets-eu.researchsquare.com/files/rs-284658/v1/0fcf975c29c095501a1e0c9f.png"},{"id":6850027,"identity":"2ef0461c-9b31-4590-8309-48471b8ee012","added_by":"auto","created_at":"2021-03-11 17:06:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":381728,"visible":true,"origin":"","legend":"Sequence alignment and activity analysis of the two CSN5 promoters. (a) Sequence alignment of VvCSN5 promoter (ProTS) and VpCSN5 promoter (ProBH) , the differential cis-regulatory elements (CREs) were markered with blue boxes. The red box indicates the start codon. The sequence alignment was made by ClustalX and CREs was predicted by PlantCare. (b) GUS staining identifies activities of two promoters and the 35S:GUS as a control. Three leaves were stained each group. The present results indicate at least two closely stained leaves (bars = 1cm). (c) Fluorometric quantitative analysis of GUS activity in each transformed leaves. GUS activity was analyzed fluorometrically and expressed as nmol 4-methylumbelliferone (MU)/mg protein min-1. Different letters denote statistically significant differences (P \u003c 0.05, ANOVA and post-hoc Tukey’s HSD test). ","description":"","filename":"Slide6.png","url":"https://assets-eu.researchsquare.com/files/rs-284658/v1/70fe295debee2e1a72770d58.png"},{"id":15672463,"identity":"8741fa1a-853d-4df0-b247-c46fd3838ac4","added_by":"auto","created_at":"2021-11-18 14:12:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1990145,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-284658/v1/ecf63a05-2cfa-4b9f-8be8-7cbd8a00b255.pdf"},{"id":6849415,"identity":"8ca7e4dd-f5c2-499e-8eeb-176bc9b87909","added_by":"auto","created_at":"2021-03-11 17:03:27","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1286581,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryinformationFigureS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-284658/v1/7f14ebffcad91f1c1670278b.docx"},{"id":6850025,"identity":"e83064bf-5fb2-48e2-ac35-af685f353fa1","added_by":"auto","created_at":"2021-03-11 17:06:27","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":152395,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryinformationTableS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-284658/v1/1f9e2bd3e2308bbc68b4c408.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eTransient Silence of VvCSN5 Enhances Powdery Mildew Resistance in Grapevine (\u003cem\u003eVitis Vinifera\u003c/em\u003e)\u003c/p\u003e","fulltext":[{"header":"Introduction","content":" \u003cp\u003eGrapevine is one of the most important fruit crops in the world, with over 7000 years cultivation history (Jaillon et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Among all grapevine species, Eurasian grapevine species \u003cem\u003eVitis vinifera\u003c/em\u003e is the main cultivar for viticulture and wine production on account of its superior aroma and flavor characteristics. However, \u003cem\u003eV. vinifera\u003c/em\u003e is vulnerable to various diseases, including powdery mildew caused by an obligate biotrophic fungus \u003cem\u003eErysiphe necator\u003c/em\u003e, which infects all green tissues of grapevine, causes a widespread and devastating disease (Gadoury et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Qiu et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In contrast, several wild Chinese grapevines species have a strong powdery mildew resistance, such as \u003cem\u003eVitis pseudoreticulata\u003c/em\u003e accession Baihe-35-1 (Gao et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Hu et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), which is therefore an important germplasm for enhancing powdery mildew resistance of \u003cem\u003eV. vinifera\u003c/em\u003e through genetic improvement.\u003c/p\u003e \u003cp\u003eTo date, several powdery mildew resistance gene loci, including \u003cem\u003eRUN1\u003c/em\u003e, \u003cem\u003eRUN2\u003c/em\u003e, \u003cem\u003eREN5\u003c/em\u003e in \u003cem\u003eMuscadinia rotundifolia\u003c/em\u003e (Barker et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Riaz et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Blanc et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), and \u003cem\u003eREN4\u003c/em\u003e, \u003cem\u003eREN6\u003c/em\u003e, \u003cem\u003eREN7\u003c/em\u003e in \u003cem\u003eV. romanetii\u003c/em\u003e and \u003cem\u003eV. piasezkii\u003c/em\u003e (Ramming et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Pap et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Mahanil et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) had been identified through forward genetic approaches. However, the molecular mechanisms research of grapevine resistance (R) genes is difficult because of its characteristic of perennial woody plants with a heterozygous genome (Feechan et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePlants have an innate immune system to avoid pathogen infection, and the two major branches of which are PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI) (Jones and Dangl \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The first branch of PTI uses transmembrane pattern recognition receptors (PRRs) to recognize multiple conserved microbial- or pathogen-associated molecular patterns (MAMPs and PAMPs) and cause penetration resistance eventually (Zipfel and Felix \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The second branch of ETI relies on a large amount of nucleotide-binding leucine-rich-repeat (NB-LRR) proteins encoded by R-genes, which can identify the effectors released from a pathogen and then lead to hypersensitive response (HR) (Jones and Dangl \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Dangl and Jones \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). As described above, the powdery mildew resistance in grapevine is also composed of penetration resistance and hypersensitive response (Douchkov et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). These two processes are interacted and impede the growth of powdery mildew by cell wall deposition and programmed cell death (Jones and Dangl \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Boutrot and Zipfel \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Hu et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCOP9 signalosome complex subunit 5 (CSN5) is one of the eight subunits of COP9 (constitutive photomorphogenesis 9) signalosome (CSN) in \u003cem\u003eArabidopsis\u003c/em\u003e. It participated in regulating CULLIN-RING E3 ubiquitin ligases (CRLs) activity as the catalytic center of the complex (Wei and Deng \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Cope et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Gusmaroli et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). As the most conservative subunit in CSN, CSN5 has become a common target of pathogenic effectors (Echalier et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Jin et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In \u003cem\u003eArabidopsis\u003c/em\u003e, AtCSN5a interacts with 29 distinct effectors from \u003cem\u003eHyaloperonospora arabidopsidis\u003c/em\u003e (\u003cem\u003eHpa\u003c/em\u003e) and \u003cem\u003ePseudomonas syringae\u003c/em\u003e (\u003cem\u003ePsy\u003c/em\u003e), and AtCSN5a mutations increase resistance to both \u003cem\u003eHpa\u003c/em\u003e and \u003cem\u003ePsy\u003c/em\u003e (Mukhtar et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The role of CSN5 in biotic stress responses has also been reported in tobacco (Liu et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), tomato (Shang et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), wheat (Zhang et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and rice (He et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, there is no report focus on CSN5 regulating defense response in grapevine.\u003c/p\u003e \u003cp\u003eIn the present study, we identified a grapevine gene \u003cem\u003eCSN5\u003c/em\u003e which showed different expression patterns in susceptible and resistant grapevine during powdery mildew infection. Through transient transformation, we discovered that silencing \u003cem\u003eVvCSN5\u003c/em\u003e enhanced powdery mildew resistance in susceptible cultivar \u003cem\u003eV. vinifera\u003c/em\u003e \u0026lsquo;Thomspon Seedless\u0026rsquo; leaves. Further analysis of the promoter structure and function indicated that the differences in \u003cem\u003eCSN5\u003c/em\u003e promoter between \u0026lsquo;Thomspon Seedless\u0026rsquo; and \u0026lsquo;Baihe-35-1\u0026rsquo; may be one of the reasons for their different resistance to powdery mildew. Our findings suggested that grapevine CSN5 negatively regulated powdery mildew resistance.\u003c/p\u003e "},{"header":"Materials And Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and growth conditions\u003c/h2\u003e \u003cp\u003eThe tissue culture seedlings of \u003cem\u003eV. vinifera\u003c/em\u003e cv. Thompson Seedless and \u003cem\u003eV. pseudoreticulata\u003c/em\u003e accession Baihe-35-1 were transplanted in individual pots containing soil mix (peat: perlite: vermiculite, 4:1:1, v:v:v), and grown in a illumination incubator for two weeks with temperatures ranging from 22\u0026deg;C to 26\u0026deg;C, under a 14 h/10 h (light/dark) light cycle.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCloning of grapevine\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eCSN5\u003c/span\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe grapevine \u003cem\u003eCSN5\u003c/em\u003e genes from \u0026lsquo;Thompson Seedless\u0026rsquo; (\u003cem\u003eVvCSN5\u003c/em\u003e) and \u0026lsquo;Baihe-35-1\u0026rsquo; (\u003cem\u003eVpCSN5\u003c/em\u003e) were amplified from corresponding leaves cDNA by Planta Max Super-Fidelity DNA Polymerase (Vazyme Bio Co., Nanjing, China), respectively. The amplified primers were derived from the \u003cem\u003eCSN5\u003c/em\u003e gene sequence we uploaded to the National Center for Biotechnology Information (NCBI) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/Blast.cgi\u003c/span\u003e\u003c/span\u003e). The PCR products were cloned into the pMD19-T vector (Takara Bio Inc., Dalian, China) and sequenced at the Beijing AuGCT Biotech, Yangling, Sequencing Department, then aligned with the genome of \u003cem\u003eV. vinifera\u003c/em\u003e PN40024 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/genome\u003c/span\u003e\u003c/span\u003e). The sequence of amplified primers and related GeneBank accession number were shown in Table S1 and Table S2, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePlasmid construction\u003c/h2\u003e \u003cp\u003eTo obtain over-expression recombinant vector, full length \u003cem\u003eVpCSN5\u003c/em\u003e gene was amplified from the pMD19-T-VpCSN5 fusion plasmid, and inserted into \u003cem\u003eBam\u003c/em\u003eHI and \u003cem\u003eKpn\u003c/em\u003eI sites in the pCAMBIA2300 vector containing the CaMV 35S promoter and green fluorescent protein (GFP) coding sequence by homologous recombination to generate 35S::VpCSN5-GFP.\u003c/p\u003e \u003cp\u003eTo silence \u003cem\u003eVvCSN5\u003c/em\u003e in \u0026lsquo;Thompson Seedless\u0026rsquo;, the vector pK7WIWG2D was used as the RNA interference vector. A 358 bp length fragment from pMD19-T-VvCSN5 was selected and amplified for constructing pK7WIWG2D\u003cem\u003e-VvCSN5\u003c/em\u003e vector through Invitrogen Gateway recombination cloning technology (Thermo Fisher Inc., Carlsbad, USA).\u003c/p\u003e \u003cp\u003eTo analyze the promoters of \u003cem\u003eVvCSN5\u003c/em\u003e and \u003cem\u003eVpCSN5\u003c/em\u003e, we designed the primers according to the genome of \u003cem\u003eV. vinifera\u003c/em\u003e PN40024, and amplified about 1500 bp promoter sequences upstream the initiation codon of \u003cem\u003eCSN5\u003c/em\u003e gene from \u0026lsquo;Thompson Seedless\u0026rsquo; and \u0026lsquo;Baihe-35-1\u0026rsquo;, respectively. The PCR products were then cloned into the pMD19-T vector and sequenced. To construct the reporter plasmids for GUS activity analysis, we cloned the \u003cem\u003eVvCSN5\u003c/em\u003e promoter (ProTS) and \u003cem\u003eVpCSN5\u003c/em\u003e promoter (ProBH) sequences into pBI121 vector to drive encoding β-glucuronidase (GUS) gene, generated construct \u003cem\u003eProTS:GUS\u003c/em\u003e and \u003cem\u003eProBH:GUS\u003c/em\u003e. The pBI121 vector was pre-digested by \u003cem\u003eHind\u003c/em\u003eIII and \u003cem\u003eBam\u003c/em\u003eHI enzyme. The sequence of related primers was shown in Table S1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSubcellular location assay\u003c/h2\u003e \u003cp\u003eTo explore the subcellular localization of grapevine CSN5, we used the 35S::VpCSN5-GFP plasmid that mentioned above, and transiently expressed in grapevine mesophyll protoplasts was performed as previous study (Zhao et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), the \u003cem\u003e35S:GFP\u003c/em\u003e as a control. The transformed protoplasts were incubated for 20\u0026ndash;25 h at room temperature in the dark, then observed by Olympus BX51 fluorescence microscopy (Japan).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eAgrobacterium\u003c/span\u003e \u003cb\u003e-mediated transient transformation and inoculation\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101 was used for the transient transformation of related plasmids. Firstly, centrifuged the shaken bacterial solution (OD600, 0.5\u0026ndash;0.6) and resuspensed it with an equal volume of induction buffer (3mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 50mM MES (pH 5.6), 0.5% (w/v) Glucose, 100\u0026micro;M Acetosyringone), 28\u0026deg;C, 180 rpm shaking for one hour. Then, chose the third or fourth fully expanded leaves, carefully injected the suspending liquid into back of leaves with a disposable sterile syringe. The method of transient transformation referred to previous studies (Kurth et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Krenek et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Bertazzon et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Urso et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Xie et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Three days later, transformed leaves were inoculated with fresh conidia of powdery mildew isolate \u003cem\u003eEn\u003c/em\u003e NAFU1, which through touching the epidermis of the transformed leaves using heavily infected \u0026lsquo;Thompson Seedless\u0026rsquo; leaves (Gao et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of resistance to powdery mildew\u003c/h2\u003e \u003cp\u003eThree groups of transformed leaves, \u003cem\u003epK7WIWG2D-VvCSN5\u003c/em\u003e (RNAi), \u003cem\u003e35S::VpCSN5-GFP\u003c/em\u003e (OE) and \u003cem\u003e35S::GFP\u003c/em\u003e (as a control, EV) were assessed for resistance to \u003cem\u003eEn\u003c/em\u003e NAFU1 (Gao et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). 3,3\u0026rsquo;-diaminobenzidine (DAB) and trypan blue (TB) stained the epidermal cells of transformed leaves were observed at 2 and 5 days post inoculation (dpi) to identify the accumulation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, fungal structures and dead host cells. Aniline blue-staining was used to visualize the accumulation of callose at 5 dpi (Gao et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Hu et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wan et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). All the samples were then examined using an Olympus BX51 microscope (Japan). Hyphal length of \u003cem\u003eEn\u003c/em\u003e NAFU1 and frequencies of the invasion induced hypersensitive cell death (full of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) were directly measured under Olympus BX51 using the CellSens operation software (Hu et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time PCR (RT-qPCR) analysis\u003c/h2\u003e \u003cp\u003eTo explore the expression patterns of \u003cem\u003eVvCSN5\u003c/em\u003e and \u003cem\u003eVpCSN5\u003c/em\u003e during the powdery mildew infection, we selected disease-free leaves from \u0026lsquo;Thompson Seedless\u0026rsquo; and \u0026lsquo;Baihe-35-1\u0026rsquo; for powdery mildew isolate \u003cem\u003eEn\u003c/em\u003e NAFU1 inoculation. Then collected leaves with 0, 12, 24, 48, 72, 96, 120 hours post inoculation (hpi) in turn, samples were promptly stored in -80℃ refrigerator after quick freezing by liquid nitrogen. The sampling method of RT-qPCR for defense-related genes was the same as above.\u003c/p\u003e \u003cp\u003eTotal RNA was extracted from collected leaves using E.Z.N.A. Plant RNA Kit (Omega, Guangzhou, China). The cDNA was obtained by reverse transcription using HiScript Q Select RT SuperMix (Vazyme, Nanjing, China). RT-qRCR assays were refer to previous study (Zhang et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Grapevine \u003cem\u003eACTIN7\u003c/em\u003e (GenBank accession number is XM_002282480.4) was used as an internal control (Wang et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Gutha et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Reid et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The relative transcript levels of the genes were calculated using the 2\u003csup\u003e\u0026minus;△△Ct\u003c/sup\u003e and Normalized Expression method. Each sample was analyzed in three biological replications. The statistical significance was evaluated by Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test. The sequence of RT-qPCR primers and gene accession number were shown in Table S1 and S2, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHistochemical and fluorometric assays for GUS activity\u003c/h2\u003e \u003cp\u003eThe histochemical GUS assay of leaves was carried out as previously described (Jefferson \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). The \u003cem\u003eVvCSN5\u003c/em\u003e promoter:\u003cem\u003eGUS\u003c/em\u003e (\u003cem\u003eProTS:GUS\u003c/em\u003e) and \u003cem\u003eVpCSN5\u003c/em\u003e promoter:\u003cem\u003eGUS\u003c/em\u003e (\u003cem\u003eProBH:GUS\u003c/em\u003e) vector were transformed into fully unfolded healthy \u0026lsquo;Thomspon Seedless\u0026rsquo; or tobacco leaves by vacuumizing and \u003cem\u003e35S:GUS\u003c/em\u003e was used as a control. Three days after transformation, the transformed leaves were inoculated s by spraying the \u003cem\u003eEn\u003c/em\u003e NAFU1 spore suspension with a concentration of 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e sporangia ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Mock-inoculated leaves were sprayed with sterile water only. Collected 2 dpi leaves, placed in GUS dye and incubated at 37℃ for 24 hours. Then, 70% and 100% ethanol were successively used for decoloring at 37℃ for 10 hours. The method of GUS quantitative analysis was referred to previous study (Yu et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe relevant data were analyzed through Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test or Tukey\u0026rsquo;s HSD test using the statistics software tool IBM SPSS v26.0.0. The mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of the mean (SD) was calculated based on the results of at least three independent experiments, and significant differences compared with controls are represented by p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":" \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatics analysis and subcellular localization of grapevine CSN5\u003c/h2\u003e \u003cp\u003eAlthough there are two \u003cem\u003eCSN5\u003c/em\u003e homologous genes in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (\u003cem\u003eAtCSN5a\u003c/em\u003e and \u003cem\u003eAtCSN5b\u003c/em\u003e), only one \u003cem\u003eCSN5\u003c/em\u003e gene in \u003cem\u003eV. vinifera\u003c/em\u003e genome (Jin et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), we named \u003cem\u003eVvCSN5\u003c/em\u003e. It's located on chromosome 3, the full length of genomic DNA is 8398 bp with six exons and five introns (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eTo understand the association of CSN5 in various plants, we firstly chosen nine homologous CSN5 proteins from seven species which functions had been well studied, and used them to build a phylogenetic tree with VvCSN5 from \u0026lsquo;Thompson Seedless\u0026rsquo; and VpCSN5 from \u0026lsquo;Baihe-35-1\u0026rsquo;. We found VvCSN5 and VpCSN5 were highly homologous with their homologous proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). VvCSN5, VpCSN5 and other CSN5 proteins were also highly conserved at their functional region (MPN domain) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). These results indicated that grapevine \u003cem\u003eCSN5\u003c/em\u003e may play similar roles to those homologous genes.\u003c/p\u003e \u003cp\u003eIt was reported that CSN5 is localized in cytoplasm and nucleus in most plants (Li et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In order to explore the intracellular localization of grapevine CSN5, we transiently expressed VpCSN5-GFP fusion protein in \u0026lsquo;Baihe-35-1\u0026rsquo; grapevine mesophyll protoplasts. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, the GFP expressed from the control construct was dispersed throughout the whole cell, while VpCSN5-GFP fusion protein localized to both cytoplasm and nucleus.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eThe expression patterns of VpCSN5 and VvCSN5 are different during powdery mildew infection\u003c/h2\u003e \u003cp\u003eTo determine whether there is any difference in expression patterns of \u003cem\u003eVvCSN5\u003c/em\u003e and \u003cem\u003eVpCSN5\u003c/em\u003e after powdery mildew infection, we inoculated the susceptible grapevine \u0026lsquo;Thompson Seedless\u0026rsquo; and resistant grapevine \u0026lsquo;Baihe-35-1\u0026rsquo; using isolate \u003cem\u003eEn\u003c/em\u003e NAFU1, respectively. RT-qPCR results showed that the \u003cem\u003eVvCSN5\u003c/em\u003e transcript lightly increased in abundance and reached a peak of ~\u0026thinsp;1.4-fold at 24 hpi, while \u003cem\u003eVpCSN5\u003c/em\u003e transcript decreased rapidly at 24 hpi (0.4-fold) and then returned to a normal level at 48 hpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This suggested that grapevine CSN5 may be involved in the defense responses to powdery mildew.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTransient silence of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eVvCSN5\u003c/span\u003e \u003cb\u003eenhances resistance to powdery mildew isolate\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eEn\u003c/span\u003e \u003cb\u003eNAFU1 in grapevine\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs above mentioned, we have known that the expression patterns of CSN5 in susceptible and resistant grapevines were discrepant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Whereupon, we wondered whether altering the expression levels of \u003cem\u003eCSN5\u003c/em\u003e in \u0026lsquo;Thompson Seedless\u0026rsquo; leaves could affect their resistance to powdery mildew. We thus obtained VvCSN5-RNAi (RNAi) and 35S::VpCSN5-GFP (OE) transient transformed leaves through injection, construct \u003cem\u003e35S::GFP\u003c/em\u003e (EV) was injected as control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, most epidermal cells of injection range of \u0026lsquo;Thompson Seedless\u0026rsquo; leaf for three constructs injected showed clear GFP signals. Similarly, the western blot analysis of GFP (\u0026sim;29.9 kDa) or VpCSN5-GFP fusion protein (\u0026sim;70.2 kDa) also confirmed the normal expression of related GFP protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), which means that the transient transformation was successful. Subsequently, we detected the transcript levels of \u003cem\u003eCSN5\u003c/em\u003e in each transformed leaf or uninjected leaf (as control) by RT-qPCR. The results showed that \u003cem\u003eCSN5\u003c/em\u003e transcript levels were up-regulated over 20-fold in OE leaves and down-regulated to 0.5-fold in RNAi leaves compared with control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eTo assess the resistance to powdery mildew in leaves with different CSN5 levels,we collected transformed \u0026lsquo;Thompson Seedless\u0026rsquo; leaves stained with trypan blue (TB) and 3,3\u0026rsquo;-diaminobenzidine (DAB), and measured the total hyphal length from isolated fungal colonies. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and b, the total hyphal length per colony in the leaves of RNAi was \u0026sim;600 \u0026micro;m at 2 dpi, while the length in the leaves of OE and EV were \u0026sim;950 and 840 \u0026micro;m, respectively. Moreover, at 5 dpi, nearly 20% of invaded cells showed whole-cell H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-staining in RNAi leaves, while both the leaves of EV and OE were less than 5% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and c). In addition, cell wall deposition by callose accumulation is also an important indicator of \u003cem\u003eVitis\u003c/em\u003e resistance to powdery mildew (Consonni et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The aniline blue-staining results showed that around the necrotic epidermal cells, major callose accumulated in RNAi leaves at 5 dpi. In contrast, there was no obviously callose accumulated in the leaves of EV and OE (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). These results suggested that silencing \u003cem\u003eVvCSN5\u003c/em\u003e could enhance the resistance to powdery mildew in grapevine.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eGrapevine CSN5 involve in multiple defenses signaling pathways\u003c/h2\u003e \u003cp\u003eTo investigate which defense-related genes were affected by CSN5, we selected several typical defense-related genes to analyze their expression patterns in transformed leaves of \u0026lsquo;Thompson Seedless\u0026rsquo;. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, for SA-associated genes, \u003cem\u003eVvPR1\u003c/em\u003e (pathogenesis-related gene 1) showed a higher expression levels (over 2-fold) in RNAi leaves than that in EV at 0 dpi and remained high levels at 2 and 5 dpi, while the \u003cem\u003eVvPR1\u003c/em\u003e level in OE was lower (~\u0026thinsp;0.5-fold) than EV in each time point. \u003cem\u003eVvPR3\u003c/em\u003e (pathogenesis-related gene 3) and \u003cem\u003eVvPAD4\u003c/em\u003e (phytoalexin deficient 4) transcript levels in RNAi leaves was both slightly higher (~\u0026thinsp;1.5-fold) than that in EV at 0 dpi, except that \u003cem\u003eVvPR3\u003c/em\u003e levels at 5 dpi was not obviously differ from EV. However, the JA-associated gene \u003cem\u003eVvJAR1\u003c/em\u003e (jasmonate resistant 1) showed a stronger expression (1.3\u0026thinsp;~\u0026thinsp;2.5-fold) in OE leaves than that in EV and RNAi. For hydrogen peroxide-associated gene \u003cem\u003eVvRBOHD\u003c/em\u003e (respiratory burst oxidase homologue D), the increasing expression trend between RNAi and EV was similar, but the transcript levels at 0, 2, 5 dpi in RNAi was all significantly higher (1.5\u0026thinsp;~\u0026thinsp;2-fold) than that in EV, which implied hydrogen peroxide-pathway had a stronger activation in RNAi leaves. In addition, the ethylene-associated gene \u003cem\u003eVvACS2\u003c/em\u003e (1-amino-cyclopropane-1-carboxylate synthase 2) transcript level in RNAi was lightly higher than EV at 2 dpi. These results indicated that grapevine CSN5 was associated with multiple defense-related genes during powdery mildew infection.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe promoters of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eVvCSN5\u003c/span\u003e \u003cb\u003eand\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eVpCSN5\u003c/span\u003e \u003cb\u003eare different in structure and activity\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAccording to the sequence alignment, we found there are only two different amino acid residues between VvCSN5 and VpCSN5 (Fig. S1). Thus, we wonder whether the different expression patterns between VvCSN5 and VpCSN5 due to the differences of their promoters. We firstly compared the promoters of \u003cem\u003eVvCSN5\u003c/em\u003e (ProTS) and \u003cem\u003eVpCSN5\u003c/em\u003e (ProBH) by sequence alignment. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, there were masses of differences between these two promoter sequences, including a number of base pair substitutions and a 132 bp fragment deletion in the \u003cem\u003eVpCSN5\u003c/em\u003e promoter. To further analyze these two sequences, we forecasted the cis-regulatory elements (CREs) of the two promoters by PlantCare (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003c/span\u003e) and markered out the discrepant elements (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Some of them, such as MYB-, MYC- and ABI3-elements, had been reported to be responsive to various stresses (Dubos et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Abe et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Tamminen et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2001\u003c/span\u003e)..\u003c/p\u003e \u003cp\u003eTo verified the function of promoters, we used histochemical staining and fluorometric assays to examine expression of the GUS genes which respectively driven by ProTS or ProBH after inoculation with powdery mildew isolate \u003cem\u003eEn\u003c/em\u003e NAFU1. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb and c, the \u003cem\u003e35S:GUS\u003c/em\u003e control leaves were strongly stained by GUS dye, and the quantitative GUS assay showed similar activities in \u003cem\u003eEn\u003c/em\u003e NAFU1-inoculated and mock conditions. Furthermore, the \u003cem\u003eProTS:GUS\u003c/em\u003e in mock condition showed a very weak expression, while in \u003cem\u003eEn\u003c/em\u003e NAFU1-inoculated leaves showed a much strong activity which was 3.5-fold higher than in mock (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb and c). However, there was no significant difference in \u003cem\u003eProBH:GUS\u003c/em\u003e activity between mock and \u003cem\u003eEn\u003c/em\u003e NAFU1-inoculated leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb and c). We also expressed \u003cem\u003eProTS:GUS\u003c/em\u003e and \u003cem\u003eProBH:GUS\u003c/em\u003e in tobacco leaves (for a higher transformation efficiency) with mock condition to confirm the reliability of GUS staining results of grapevine leaves. We discovered stronger staining in tobacco leaves than that in corresponding grapevine leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), and similar to mock in grapevine leaves, the \u003cem\u003eProBH:GUS\u003c/em\u003e activity in tobacco was also higher than \u003cem\u003eProTS:GUS\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). These results implied that the differences in expression patterns of CSN5 between susceptible and resistant grapevine may cause by different promoter activation.\u003c/p\u003e \u003c/div\u003e "},{"header":"Discussion","content":" \u003cp\u003eAs the catalytic center of COP9 signalosome (CSN), CSN5 plays an essential role for normal working of complex (Stratmann and Gusmaroli \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Jin et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Thus, CSN5 is quite conserved in eukaryotes (Cope et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). We already know that CSN5 is involved in the process of pathogen defense in many plants, including \u003cem\u003eArabidopsis\u003c/em\u003e (Mukhtar et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), tobacco (Liu et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), wheat (Zhang et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), rice (He et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), etc. However, there was no related research on grapevine CSN5. In this study, we demonstrated the conservation of CSN5, and further discovered that it was associated with grapevine resistance to powdery mildew.\u003c/p\u003e \u003cp\u003eAccording to the expression analysis of grapevine CSN5 during powdery mildew infection, we found that the \u003cem\u003eVpCSN5\u003c/em\u003e expression reached a minimum at 24 hpi, whereas \u003cem\u003eVvCSN5\u003c/em\u003e reached a maximum at 24 hpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), which was consistent with the timing of haustorial formation (Hu et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This result implies that the changes of CSN5 expression are pathogen induced. Further combining with the results of stronger powdery mildew resistance of CSN5-silenced leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), which both supporting a negative role of CSN5 on powdery mildew infection.\u003c/p\u003e \u003cp\u003ePrevious studies showed CSN5 was mainly involved in plant pathogen-defense through JA- or SA-signaling pathways (Feng et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Hind et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Spoel et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Interestingly, the effects of CSN5 to the two pathways are opposite. For example, the over-expression of \u003cem\u003eOsCSN5a\u003c/em\u003e inhibits rice black-streaked dwarf virus (RBSDV) infection through activating JA-signaling pathway in rice (He et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Whereas silencing \u003cem\u003eTaCSN5\u003c/em\u003e in wheat improves wheat leaf rust resistance by activation of SA-signaling pathway (Zhang et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In our study, a stronger powdery mildew resistance showed in CSN5-RNAi leaves and three SA-associated marker genes (\u003cem\u003eVvPR1\u003c/em\u003e, \u003cem\u003eVvPR3\u003c/em\u003e, \u003cem\u003eVvPAD4\u003c/em\u003e) had higher basal expression levels (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), which suggested that the role of grapevine CSN5 in pathogen defense is probably similar to \u003cem\u003eTaCSN5\u003c/em\u003e in wheat. In addition, the JA-associated marker gene \u003cem\u003eVvJAR1\u003c/em\u003e showed stronger expression in CSN5 over-expression leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), which suggested grapevine CSN5 may be positively correlated with JA-signaling pathway, like its homologous genes in \u003cem\u003eArabidopsis\u003c/em\u003e (Feng et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInterestingly, there is only one different amino acid residue in the MPN domain of VvCSN5 and VpCSN5 (Fig. S1), however, there were significant differences in their expression patterns during powdery mildew infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). We know that gene expression depends on its promoter, in which pathogen-inducible gene expression requires the interaction between defense-related transcription factors (TFs) and cis-regulatory elements (CREs) in the promoter (Amorim et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Rushton et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). For example, promoter mutations in rice \u003cem\u003eXa13\u003c/em\u003e gene cause down-regulation of expression during host\u0026ndash;pathogen interaction, resulting in the race-specific resistance to \u003cem\u003eXanthomonas oryzae\u003c/em\u003e pv. \u003cem\u003eOryzae\u003c/em\u003e (Chu et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In grapevine, \u0026lsquo;Baihe-35-1\u0026rsquo; \u003cem\u003eVpRFP1\u003c/em\u003e gene expressed under its own promoter activated a stronger disease resistance than driven by \u003cem\u003eV. vinifera\u003c/em\u003e cv. Carignane \u003cem\u003eVvRFP1\u003c/em\u003e promoter (Yu et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In this study, we found that there were many base pair substitutions and a 132 bp fragment deletion in \u003cem\u003eVpCSN5\u003c/em\u003e promoter that resulted in more CAAT-box and stress-related CREs existed in \u003cem\u003eVvCSN5\u003c/em\u003e promoter sequence than that in \u003cem\u003eVpCSN5\u003c/em\u003e promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), including MYB-elements and ERE-elements, which were reported to be responsive to salicylic acid and ethylene (Yang and Klessig \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Fujimoto et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), respectively. This explained the reason of \u003cem\u003eVvCSN5\u003c/em\u003e promoter displayed a strong inducible GUS activity in response to powdery mildew (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb and c).\u003c/p\u003e \u003cp\u003eOur study expounded the effect of CSN5 on grapevine resistance to powdery mildew, confirmed the functional consistence between grapevine \u003cem\u003eCSN5\u003c/em\u003e and its homologous genes. Further demonstrate the different structures of \u003cem\u003eCSN5\u003c/em\u003e promoters between susceptible and resistant grapevines caused \u003cem\u003eCSN5\u003c/em\u003e gene differently expressed during powdery mildew infection. Taken together, our results suggested grapevine \u003cem\u003eCSN5\u003c/em\u003e was a negative regulator gene for grapevine powdery mildew resistance.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCKC and YQW conceived and designed the research. CKC and ML conducted most of the experiments. GHK, XYZ, BM and YH participated in the experiments. CKC wrote the manuscript. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the National Natural Science Foundation of China (Grant No. 31972986 to YQW).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with ethical standards \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e The authors declare that they have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003eAbe H, Urao T, Ito T, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell 15(1):63\u0026ndash;78. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1105/tpc.006130\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eAmorim LLB, dos Santos RD, Neto JPB, Guida-Santos M, Crovella S, Benko-Iseppon AM (2017) Transcription Factors Involved in Plant Resistance to Pathogens. Curr Protein Pept Sc 18(4):335\u0026ndash;351. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2174/1389203717666160619185308\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eBarker CL, Donald T, Pauquet J, Ratnaparkhe MB, Bouquet A, Adam-Blondon AF, Thomas MR, Dry I (2005) Genetic and physical mapping of the grapevine powdery mildew resistance gene, Run1, using a bacterial artificial chromosome library. TAG Theoretical and applied genetics Theoretische und angewandte Genetik 111 (2):370\u0026ndash;377. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00122-005-2030-8\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eBertazzon N, Raiola A, Castiglioni C, Gardiman M, Angelini E, Borgo M, Ferrari S (2012) Transient silencing of the grapevine gene VvPGIP1 by agroinfiltration with a construct for RNA interference. Plant Cell Rep 31(1):133\u0026ndash;143. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00299-011-1147-2\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eBlanc S, Wiedemann-Merdinoglu S, Dumas V, Mestre P, Merdinoglu D (2012) A reference genetic map of Muscadinia rotundifolia and identification of Ren5, a new major locus for resistance to grapevine powdery mildew. TAG Theoretical and applied genetics Theoretische und angewandte Genetik 125 (8):1663\u0026ndash;1675. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00122-012-1942-3\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eBoutrot F, Zipfel C (2017) Function, Discovery, and Exploitation of Plant Pattern Recognition Receptors for Broad-Spectrum Disease Resistance. Annu Rev Phytopathol 55:257\u0026ndash;286. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1146/annurev-phyto-080614-120106\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eChu Z, Yuan M, Yao J, Ge X, Yuan B, Xu C, Li X, Fu B, Li Z, Bennetzen JL, Zhang Q, Wang S (2006) Promoter mutations of an essential gene for pollen development result in disease resistance in rice. Genes Dev 20(10):1250\u0026ndash;1255. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1101/gad.1416306\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eConsonni C, Humphry ME, Hartmann HA, Livaja M, Durner J, Westphal L, Vogel J, Lipka V, Kemmerling B, Schulze-Lefert P, Somerville SC, Panstruga R (2006) Conserved requirement for a plant host cell protein in powdery mildew pathogenesis. Nat Genet 38(6):716\u0026ndash;720. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/ng1806\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eCope GA, Suh GS, Aravind L, Schwarz SE, Zipursky SL, Koonin EV, Deshaies RJ (2002) Role of predicted metalloprotease motif of Jab1/Csn5 in cleavage of Nedd8 from Cul1. Science 298(5593):608\u0026ndash;611. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/science.1075901\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eDangl JL, Jones JD (2001) Plant pathogens and integrated defence responses to infection. Nature 411(6839):826\u0026ndash;833. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/35081161\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eDouchkov D, Lueck S, Hensel G, Kumlehn J, Rajaraman J, Johrde A, Doblin MS, Beahan CT, Kopischke M, Fuchs R, Lipka V, Niks RE, Bulone V, Chowdhury J, Little A, Burton RA, Bacic A, Fincher GB, Schweizer P (2016) The barley (Hordeum vulgare) cellulose synthase-like D2 gene (HvCslD2) mediates penetration resistance to host-adapted and nonhost isolates of the powdery mildew fungus. New Phytol 212(2):421\u0026ndash;433. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/nph.14065\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eDubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, Lepiniec L (2010) MYB transcription factors in Arabidopsis. Trends Plant Sci 15(10):573\u0026ndash;581. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.tplants.2010.06.005\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eEchalier A, Pan Y, Birol M, Tavernier N, Pintard L, Hoh F, Ebel C, Galophe N, Claret FX, Dumas C (2013) Insights into the regulation of the human COP9 signalosome catalytic subunit, CSN5/Jab1. Proc Natl Acad Sci U S A 110(4):1273\u0026ndash;1278. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.1209345110\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eFeechan A, Anderson C, Torregrosa L, Jermakow A, Mestre P, Wiedemann-Merdinoglu S, Merdinoglu D, Walker AR, Cadle-Davidson L, Reisch B, Aubourg S, Bentahar N, Shrestha B, Bouquet A, Adam-Blondon AF, Thomas MR, Dry IB (2013) Genetic dissection of a TIR-NB-LRR locus from the wild North American grapevine species Muscadinia rotundifolia identifies paralogous genes conferring resistance to major fungal and oomycete pathogens in cultivated grapevine. Plant J 76(4):661\u0026ndash;674. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/tpj.12327\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eFeng S, Ma L, Wang X, Xie D, Dinesh-Kumar SP, Wei N, Deng XW (2003) The COP9 signalosome interacts physically with SCF COI1 and modulates jasmonate responses. Plant Cell 15(5):1083\u0026ndash;1094. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1105/tpc.010207\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eFujimoto SY, Ohta M, Usui A, Shinshi H, Ohme-Takagi M (2000) Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC box-mediated gene expression. Plant Cell 12(3):393\u0026ndash;404. doi:Doi \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2307/3870944\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eGadoury DM, Cadle-Davidson L, Wilcox WF, Dry IB, Seem RC, Milgroom MG (2012) Grapevine powdery mildew (Erysiphe necator): a fascinating system for the study of the biology, ecology and epidemiology of an obligate biotroph. Mol Plant Pathol 13(1):1\u0026ndash;16. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1364-3703.2011.00728.x\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eGao YR, Han YT, Zhao FL, Li YJ, Cheng Y, Ding Q, Wang YJ, Wen YQ (2016) Identification and utilization of a new Erysiphe necator isolate NAFU1 to quickly evaluate powdery mildew resistance in wild Chinese grapevine species using detached leaves. Plant physiology biochemistry: PPB 98:12\u0026ndash;24. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.plaphy.2015.11.003\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eGusmaroli G, Figueroa P, Serino G, Deng XW (2007) Role of the MPN subunits in COP9 signalosome assembly and activity, and their regulatory interaction with Arabidopsis cullin3-based E3 ligases. Plant Cell 19(2):564\u0026ndash;581. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1105/tpc.106.047571\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eGutha LR, Casassa LF, Harbertson JF, Naidu RA (2010) Modulation of flavonoid biosynthetic pathway genes and anthocyanins due to virus infection in grapevine (Vitis vinifera L.) leaves. Bmc Plant Biology 10. doi:Artn 187\u003c/p\u003e\n\u003cp\u003e1186/1471-2229-10-187\u003c/p\u003e\n\u003cp\u003eHe L, Chen X, Yang J, Zhang T, Li J, Zhang S, Zhong K, Zhang H, Chen J, Yang J (2020) Rice black-streaked dwarf virus-encoded P5-1 regulates the ubiquitination activity of SCF E3 ligases and inhibits jasmonate signaling to benefit its infection in rice. New Phytol 225(2):896\u0026ndash;912. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/nph.16066\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eHind SR, Pulliam SE, Veronese P, Shantharaj D, Nazir A, Jacobs NS, Stratmann JW (2011) The COP9 signalosome controls jasmonic acid synthesis and plant responses to herbivory and pathogens. Plant J 65(3):480\u0026ndash;491. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1365-313X.2010.04437.x\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eHu Y, Gao YR, Yang LS, Wang W, Wang YJ, Wen YQ (2019) The cytological basis of powdery mildew resistance in wild Chinese Vitis species. Plant Physiol Biochem 144:244\u0026ndash;253. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.plaphy.2019.09.049\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eHu Y, Li Y, Hou F, Wan D, Cheng Y, Han Y, Gao Y, Liu J, Guo Y, Xiao S, Wang Y, Wen YQ (2018) Ectopic expression of Arabidopsis broad-spectrum resistance gene RPW8.2 improves the resistance to powdery mildew in grapevine (Vitis vinifera). Plant Sci 267:20\u0026ndash;31. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.plantsci.2017.11.005\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eJaillon O, Aury JM, Noel B, Policriti A, Clepet C, Casagrande A, Choisne N, Aubourg S, Vitulo N, Jubin C, Vezzi A, Legeai F, Hugueney P, Dasilva C, Horner D, Mica E, Jublot D, Poulain J, Bruyere C, Billault A, Segurens B, Gouyvenoux M, Ugarte E, Cattonaro F, Anthouard V, Vico V, Del Fabbro C, Alaux M, Di Gaspero G, Dumas V, Felice N, Paillard S, Juman I, Moroldo M, Scalabrin S, Canaguier A, Le Clainche I, Malacrida G, Durand E, Pesole G, Laucou V, Chatelet P, Merdinoglu D, Delledonne M, Pezzotti M, Lecharny A, Scarpelli C, Artiguenave F, Pe ME, Valle G, Morgante M, Caboche M, Adam-Blondon AF, Weissenbach J, Quetier F, Wincker P, French-Italian Public Consortium for Grapevine Genome C (2007) The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature 449 (7161):463\u0026ndash;467. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nature06148\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eJefferson R (1987) Assaying chimeric genes in plants: the GUS Gene Fusion System. Plant Mol Biol Rep 5(4):387\u0026ndash;405\u003c/p\u003e\n\u003cp\u003eJin D, Li B, Deng XW, Wei N (2014) Plant COP9 signalosome subunit 5, CSN5. Plant Sci 224:54\u0026ndash;61. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.plantsci.2014.04.001\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eJones JD, Dangl JL (2006) The plant immune system. Nature 444(7117):323\u0026ndash;329. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nature05286\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eKrenek P, Samajova O, Luptovciak I, Doskocilova A, Komis G, Samaj J (2015) Transient plant transformation mediated by Agrobacterium tumefaciens: Principles, methods and applications. Biotechnol Adv 33(6 Pt 2):1024\u0026ndash;1042. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.biotechadv.2015.03.012\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eKurth EG, Peremyslov VV, Prokhnevsky AI, Kasschau KD, Miller M, Carrington JC, Dolja VV (2012) Virus-derived gene expression and RNA interference vector for grapevine. J Virol 86(11):6002\u0026ndash;6009. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/JVI.00436-12\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eLi Y, Chu ZN, Luo JY, Zhou YH, Cai YJ, Lu YG, Xia JH, Kuang HH, Ye ZB, Ouyang B (2018) The C2H2 zinc-finger protein SlZF3 regulates AsA synthesis and salt tolerance by interacting with CSN5B. Plant Biotechnol J 16(6):1201\u0026ndash;1213. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/pbi.12863\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eLiu J, Zhao FL, Guo Y, Fan XC, Wang YJ, Wen YQJPC, Tissue, Culture O (2019) The ABA receptor-like gene VyPYL9 from drought-resistance wild grapevine confers drought tolerance and ABA hypersensitivity in Arabidopsis. 138 (3):543\u0026ndash;558\u003c/p\u003e\n\u003cp\u003eLiu Y, Schiff M, Serino G, Deng XW, Dinesh-Kumar SP (2002) Role of SCF ubiquitin-ligase and the COP9 signalosome in the N gene-mediated resistance response to Tobacco mosaic virus. Plant Cell 14(7):1483\u0026ndash;1496. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1105/tpc.002493\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eMahanil S, Ramming D, Cadle-Davidson M, Owens C, Garris A, Myles S, Cadle-Davidson L (2012) Development of marker sets useful in the early selection of Ren4 powdery mildew resistance and seedlessness for table and raisin grape breeding. TAG Theoretical and applied genetics Theoretische und angewandte Genetik 124 (1):23\u0026ndash;33. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00122-011-1684-7\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eMukhtar MS, Carvunis AR, Dreze M, Epple P, Steinbrenner J, Moore J, Tasan M, Galli M, Hao T, Nishimura MT, Pevzner SJ, Donovan SE, Ghamsari L, Santhanam B, Romero V, Poulin MM, Gebreab F, Gutierrez BJ, Tam S, Monachello D, Boxem M, Harbort CJ, McDonald N, Gai L, Chen H, He Y, European U, Effectoromics C, Vandenhaute J, Roth FP, Hill DE, Ecker JR, Vidal M, Beynon J, Braun P, Dangl JL (2011) Independently evolved virulence effectors converge onto hubs in a plant immune system network. Science 333(6042):596\u0026ndash;601. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/science.1203659\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003ePap D, Riaz S, Dry IB, Jermakow A, Tenscher AC, Cantu D, Olah R, Walker MA (2016) Identification of two novel powdery mildew resistance loci, Ren6 and Ren7, from the wild Chinese grape species Vitis piasezkii. BMC Plant Biol 16(1):170. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12870-016-0855-8\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eQiu W, Feechan A, Dry I (2015) Current understanding of grapevine defense mechanisms against the biotrophic fungus (Erysiphe necator), the causal agent of powdery mildew disease. Hortic Res 2:15020. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/hortres.2015.20\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eRamming DW, Gabler F, Smilanick J, Cadle-Davidson M, Barba P, Mahanil S, Cadle-Davidson L (2011) A single dominant locus, ren4, confers rapid non-race-specific resistance to grapevine powdery mildew. Phytopathology 101(4):502\u0026ndash;508. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1094/PHYTO-09-10-0237\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eReid KE, Olsson N, Schlosser J, Peng F, Lund ST (2006) An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development. Bmc Plant Biology 6. doi:Artn 2710.1186/1471-2229-6-27\u003c/p\u003e\n\u003cp\u003eRiaz S, Tenscher AC, Ramming DW, Walker MA (2011) Using a limited mapping strategy to identify major QTLs for resistance to grapevine powdery mildew (Erysiphe necator) and their use in marker-assisted breeding. TAG Theoretical and applied genetics Theoretische und angewandte Genetik 122 (6):1059\u0026ndash;1073. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00122-010-1511-6\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eRushton PJ, Reinstadler A, Lipka V, Lippok B, Somssich IE (2002) Synthetic plant promoters containing defined regulatory elements provide novel insights into pathogen- and wound-induced signaling. Plant Cell 14(4):749\u0026ndash;762. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1105/tpc.010412\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eShang Y, Wang K, Sun S, Zhou J, Yu JQ (2019) COP9 Signalosome CSN4 and CSN5 Subunits Are Involved in Jasmonate-Dependent Defense Against Root-Knot Nematode in Tomato. Front Plant Sci 10:1223. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fpls.2019.01223\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eSpoel SH, Mou Z, Tada Y, Spivey NW, Genschik P, Dong X (2009) Proteasome-mediated turnover of the transcription coactivator NPR1 plays dual roles in regulating plant immunity. Cell 137(5):860\u0026ndash;872. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cell.2009.03.038\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eStratmann JW, Gusmaroli G (2012) Many jobs for one good cop - the COP9 signalosome guards development and defense. Plant Sci 185\u0026ndash;186:50\u0026ndash;64. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.plantsci.2011.10.004\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eTamminen I, Makela P, Heino P, Palva ET (2001) Ectopic expression of ABI3 gene enhances freezing tolerance in response to abscisic acid and low temperature in Arabidopsis thaliana. Plant J 25(1):1\u0026ndash;8. doi:DOI \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1046/j.1365-313x.2001.00927.x\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eUrso S, Zottini M, Ruberti C, Lo Schiavo F, Stanca AM, Cattivelli L, Val\u0026egrave; G (2013) An Agrobacterium tumefaciens-mediated gene silencing system for functional analysis in grapevine. Plant Cell Tissue Organ Cult 114(1):49\u0026ndash;60. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s11240-013-0305-3\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eWan DY, Guo Y, Cheng Y, Hu Y, Xiao S, Wang Y, Wen YQ (2020) CRISPR/Cas9-mediated mutagenesis of VvMLO3 results in enhanced resistance to powdery mildew in grapevine (Vitis vinifera). Hortic Res 7:116. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41438-020-0339-8\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eWang J, Yao W, Wang L, Ma F, Tong W, Wang C, Bao R, Jiang C, Yang Y, Zhang J, Xu Y, Wang X, Zhang C, Wang Y (2017) Overexpression of VpEIFP1, a novel F-box/Kelch-repeat protein from wild Chinese Vitis pseudoreticulata, confers higher tolerance to powdery mildew by inducing thioredoxin z proteolysis. Plant Sci 263:142\u0026ndash;155. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.plantsci.2017.07.004\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eWang J, Yu Y, Zhang Z, Quan R, Zhang H, Ma L, Deng XW, Huang R (2013) Arabidopsis CSN5B interacts with VTC1 and modulates ascorbic acid synthesis. Plant Cell 25(2):625\u0026ndash;636. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1105/tpc.112.106880\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eWang T, Chang C, Gu C, Tang S, Xie Q, Shen QH (2016) An E3 Ligase Affects the NLR Receptor Stability and Immunity to Powdery Mildew. Plant Physiol 172(4):2504\u0026ndash;2515. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1104/pp.16.01520\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eWei N, Deng XW (2003) The COP9 signalosome. Annu Rev Cell Dev Biol 19:261\u0026ndash;286. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1146/annurev.cellbio.19.111301.112449\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eXie YG, Ma YY, Bi PP, Wei W, Liu J, Hu Y, Gou YJ, Zhu D, Wen YQ, Feng JY (2020) Transcription factor FvTCP9 promotes strawberry fruit ripening by regulating the biosynthesis of abscisic acid and anthocyanins. Plant Physiol Biochem 146:374\u0026ndash;383. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.plaphy.2019.11.004\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eYang Y, Klessig DF (1996) Isolation and characterization of a tobacco mosaic virus-inducible myb oncogene homolog from tobacco. Proc Natl Acad Sci U S A 93(25):14972\u0026ndash;14977. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.93.25.14972\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eYu Y, Xu W, Wang J, Wang L, Yao W, Xu Y, Ding J, Wang Y (2013) A core functional region of the RFP1 promoter from Chinese wild grapevine is activated by powdery mildew pathogen and heat stress. Planta 237(1):293\u0026ndash;303. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00425-012-1769-9\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eZhang H, Wang X, Giroux MJ, Huang L (2017) A wheat COP9 subunit 5-like gene is negatively involved in host response to leaf rust. Mol Plant Pathol 18(1):125\u0026ndash;133. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/mpp.12467\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eZhang K, Han YT, Zhao FL, Hu Y, Gao YR, Ma YF, Zheng Y, Wang YJ, Wen YQ (2015) Genome-wide Identification and Expression Analysis of the CDPK Gene Family in Grape, Vitis spp. BMC Plant Biol 15:164. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12870-015-0552-z\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eZhao FL, Li YJ, Hu Y, Gao YR, Zang XW, Ding Q, Wang YJ, Wen YQ (2016) A highly efficient grapevine mesophyll protoplast system for transient gene expression and the study of disease resistance proteins. Plant Cell Tiss Org 125(1):43\u0026ndash;57. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s11240-015-0928-7\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eZipfel C, Felix G (2005) Plants and animals: a different taste for microbes? Curr Opin Plant Biol 8(4):353\u0026ndash;360. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.pbi.2005.05.004\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Grapevine, Powdery mildew (Erysiphe necator), CSN5, Transient transformation, Promoter","lastPublishedDoi":"10.21203/rs.3.rs-284658/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-284658/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAs one of the most economically important fruit crops in the world, grapevine (\u003cem\u003eVitis vinifera\u003c/em\u003e) suffers significant yield losses from many pathogens including powdery mildew caused by \u003cem\u003eErysiphe necator\u003c/em\u003e. By contrast, several wild Chinese grapevines including \u003cem\u003eVitis pseudoreticulata\u003c/em\u003e accession ‘Baihe-35-1’ exhibit a high resistance to powdery mildew pathogen. Here, we identified a grapevine gene \u003cem\u003eCSN5 \u003c/em\u003e(\u003cem\u003eCOP9 signalosome complex subunit 5\u003c/em\u003e), designated \u003cem\u003eVvCSN5\u003c/em\u003e,\u003cem\u003e \u003c/em\u003ewhich showed different expression patterns in ‘Baihe-35-1’ and in susceptible cultivar \u003cem\u003eV. vinifera\u003c/em\u003e ‘Thompson Seedless’ during powdery mildew isolate \u003cem\u003eEn\u003c/em\u003e NAFU1 infection. Moreover, transient silence of \u003cem\u003eVvCSN5 \u003c/em\u003ein ‘Thompson Seedless’ leaves enhanced resistance to\u003cem\u003e En\u003c/em\u003e NAFU1, which is accompanied by cell wall deposition at the attempt sites, and hypersensitive response-like cell death of penetrated epidermal cells. Several defense-related marker genes (\u003cem\u003eVvPR1\u003c/em\u003e, \u003cem\u003eVvPR3\u003c/em\u003e, \u003cem\u003eVvPAD4\u003c/em\u003e, and \u003cem\u003eVvRBOHD\u003c/em\u003e) had higher basal expression levels in \u003cem\u003eVvCSN5\u003c/em\u003e-silenced leaves. In addition, we found the structure and activity of \u003cem\u003eCSN5\u003c/em\u003e promoters in ‘Thompson Seedless’ and ‘Baihe-35-1’ were discrepant, which may be one of the reasons for their different resistance to powdery mildew infection. Taken together, these results imply that grapevine CSN5 plays an important role in the responses to powdery mildew.\u003c/p\u003e","manuscriptTitle":"Transient Silence of VvCSN5 Enhances Powdery Mildew Resistance in Grapevine (Vitis Vinifera)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-03-11 17:03:25","doi":"10.21203/rs.3.rs-284658/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2021-03-04T00:00:00+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-03-03T00:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-03-02T00:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2021-02-27T21:33:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f0a365d4-d5e8-44f6-b1d3-e5006cc0a3fa","owner":[],"postedDate":"March 11th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":2901966,"name":"Plant Physiology and Morphology"},{"id":2901967,"name":"Plant Molecular Biology and Genetics"}],"tags":[],"updatedAt":"2021-05-02T15:51:15+00:00","versionOfRecord":[],"versionCreatedAt":"2021-03-11 17:03:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-284658","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-284658","identity":"rs-284658","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}