Characterization of caffeoyl shikimate esterase gene family identifies CsCSE5 as a positive regulator of Podosphaera xanthii and Corynespora cassiicola pathogen resistance in cucumber

CsCSE genes might be involved in the tolerance of cucumber to pathogens. Silencing of the CsCSE5 gene resulted in attenuated resistance of cucumber to Podosphaera xanthii and Corynespora cassiicola. Caffeoyl shikimate esterase (CSE), a key enzyme in the lignin biosynthetic pathway, has recently been characterized to play a key role in defense against pathogenic infection in plants. However, a systematic analysis of the CSE gene family in cucumber (Cucumis sativus) has not yet been conducted. Here, we identified eight CsCSE genes from the cucumber genome via bioinformatic analyses, and these genes were unevenly distributed on chromosomes 1, 3, 4, and 5. Results from multiple sequence alignment indicated that the CsCSE proteins had domains required for CSE activity. Phylogenetic analysis of gene structure and protein motifs revealed the conservation and diversity of the CsCSE gene family. Collinearity analysis showed that CsCSE genes had high homology with CSE genes in wax gourd (Benincasa hispida). Cis-acting element analysis of the promoters suggested that CsCSE genes might play important roles in growth, development, and stress tolerance. Expression pattern analysis indicated that CsCSE5 might be involved in regulating the resistance of cucumber to pathogens. Functional verification data confirmed that CsCSE5 positively regulates the resistance of cucumber to powdery mildew pathogen Podosphaera xanthii and target leaf spot pathogen Corynespora cassiicola. The results of our study provide information that will aid the genetic improvement of resistant cucumber varieties.


Introduction
Plants are constantly under threat from a variety of pathogens during their life cycle due to their sessility.As the first line of defense against pathogen stress, the plant cell wall plays an essential role in pathogenic stress response, which is mainly attributed to the presence of lignin, one of the key components of the cell wall (Molina et al. 2021;Xiong et al. 2021;Zhu et al. 2022).Lignin is an intricate aromatic polymer that provides mechanical strength and hydrophobicity to cell walls, facilitates water transport, and acts as a critical physical and chemical barrier in the defense response to pathogen infection (Vanholme et al. 2019;Cesarino 2019;Moura et al. 2010;Liu et al. 2018).When pathogens invade plant cells, defense-induced biosynthesis or deposition of lignin in the cell wall provides a physical barrier for the hosts to resist pathogen infection because it limits the entry of cell wall-degrading enzymes and pathogen toxins into the hosts and prevents the transmission of nutrients from the hosts to the pathogens, thereby preventing the proliferation of pathogens (Xiong et al. 2021;Dong and Lin 2021;Ma et al. 2018;Eynck et al. 2012).In addition, the accumulation of lignin can strengthen the above-mentioned defense responses and activate plant-specific immune responses by chemically modifying the cell wall, which provides a chemical barrier for hosts to resist pathogen infection (Yang et al. 2021;Engelsdorf et al. 2019;Bacete et al. 2018;Höch et al. 2021).In recent decades, numerous studies have shown that several genes involved in lignin biosynthesis contribute to the immune responses of plants to pathogens.For instance, the loss of function of broad-spectrum resistance Kitaake-1 (bsr-k1) in rice enhances the expression of phenylalanine ammonia-lyase (PAL; OsPAL1-7), which in turn improves broad-spectrum resistance to rice blast pathogen Magnaporthe oryzae and bacterial blight pathogen Xanthomonas oryzae pv.Oryzae (Zhou et al. 2018).Cotton laccase (LAC; GhLAC15) actively regulates resistance against Verticillium wilt pathogen Verticillium dahliae by mediating the accumulation of lignin and promoting defense-induced lignification in the cell wall (Zhang et al. 2019).Recently, the transcription factor GhODO1 has been shown to confer tolerance to V. dahliae in cotton by directly activating the expression of 4-coumarate:CoA ligase (4CL; Gh4CL1) and cinnamyl alcohol dehydrogenase (CAD; GhCAD3) to accelerate the accumulation of lignin (Zhu et al. 2022).In light of these key roles of lignin in defense against pathogens, the mechanism of lignin biosynthesis has been the focus of extensive research for decades.
CSE has been annotated as lysophospholipase 2 in Arabidopsis and is regarded as the central enzyme of the lignin biosynthetic pathway (Gao et al. 2010).The importance of CSE in regulating lignin biosynthesis has been demonstrated in many plants.For example, loss of function or down-regulated expression of CSE in Arabidopsis thaliana, Medicago truncatula, and two hybrid poplars (Populus alba × P. glandulosa and P. tremula × P. alba) results in a significant reduction of the lignin content (Vanholme et al. 2013;Ha et al. 2016;Jang et al. 2021;Vries et al. 2021).Overexpression of PbCSE1 promotes the lignification of pear fruits and accumulation of lignin in Arabidopsis (Xu et al. 2021).In addition, CSE has been reported to affect changes in the cell wall polysaccharide content (Saleme et al. 2017).For example, the transformation of cellulose into glucose was increased fourfold in the cell wall of a CSE deletion mutant of A. thaliana, which altered the structural composition of cell wall polysaccharides (Vargas et al. 2016).Although CSE has been isolated, cloned, and even functionally characterized in a variety of plants, few studies have examined its role in the resistance of plants to pathogen stress.
Some studies have indicated that CSE is critical for the defense responses of plants to stress; however, most studies of CSE have focused on its effects on lignocellulosic biomass (Saleme et al. 2017;Ozparpucu et al. 2019;Wang et al. 2021a, b).In Arabidopsis, CSE mutant plants show higher susceptibility to zinc and H 2 O 2 than wild-type plants, and CSE-overexpressing plants show greater resistance to cadmium and H 2 O 2 , indicating that CSE participates in the response to heavy metal stress and alleviates oxidative stress in plants (Gao et al. 2010;Wang et al. 2019).Recently, we reported for the first time that CSE plays a role in plant disease resistance; specifically, we showed that CsCSE1 positively regulates defense against powdery mildew pathogen Podosphaera xanthii attack in cucumber (Yu et al. 2022).Given that there may be similar or redundant biological functions among members of the gene family, we identified eight members of the CsCSE gene family using the complete cucumber genome sequence and annotation information.Bioinformatics analysis and expression pattern analysis revealed a candidate CsCSE gene, CsCSE5, that might be involved in cucumber-pathogen interactions, and the function of CsCSE5 was verified by virus-induced gene silencing (VIGS) technology.These findings provided new insights into the disease resistance function of CsCSE5.Our experiments will aid future studies aimed at clarifying the molecular mechanism of cucumber disease tolerance and the breeding of resistant cucumber varieties.

Identification and characterization of CsCSE genes in cucumber
We conducted manual searches of the keyword "cucumber caffeoyl shikimate esterase" through the NCBI database (https:// www.ncbi.nlm.nih.gov/) to obtain putative CsCSE genes and the corresponding protein sequences.These protein sequences were submitted to the Batch CD-search online site (https:// www.ncbi.nlm.nih.gov/ Struc ture/ bwrpsb/ bwrpsb.cgi) for further domain analysis to identify the true CsCSE genes.The physicochemical properties of CsCSE proteins, including the number of amino acids, molecular weights (MWs), isoelectric points (pIs), grand average of hydropathicity (GRAVY), and instability index were predicted using the online tool ProtParam (http:// web.expasy.org/ protp aram/).The SignalP-5.0 website (https:// servi ces.healt htech.dtu.dk/ servi ce.php?Signa lP-5.0) was used to identify the signal peptides of CsCSE proteins.

Multiple sequence alignment, gene structure, and protein motif analysis of the CsCSE family
Multiple sequence alignment of CsCSE proteins was carried out using DNAMAN software.The untranslated region (UTR), coding sequence (CDS), and intron positions of CsCSE genes were extracted from the cucumber genome annotation file (GFF format) obtained from the NCBI database and visualized using TBbtools software.The online tool MEME (http:// meme-suite.org/ tools/ meme) was used for conserved motif analysis of the CsCSE proteins with the following parameters: site distribution, zero or one occurrence per sequence; maximum number of motifs, 10; and motif width, between 6 and 50.The results were displayed using TBtools software.

Cis-acting element analysis of CsCSE promoters
To predict the putative cis-acting elements in the promoter regions of the CsCSE genes, the cucumber genome annotation file (GFF format) was used to obtain the upstream 2,000-bp sequences of the transcription start sites of the CsCSE genes, which were then input into the PlantCARE database (http:// bioin forma tics.psb.ugent.be/ webto ols/ plant care/ html/) for analysis.Finally, the obtained cis-acting elements of CsCSE were input into TBtools software for visualization.

Chromosome distribution and collinearity analysis of CsCSE genes
The chromosome position and length information of the CsCSE genes were retrieved from the cucumber genome sequence file (FNA format) and genome annotation file (GFF format).The chromosome locations of the CsCSE genes were mapped using the Advanced Circles function of Tbtools.The genome sequence file (FNA format) and genome annotation file (GFF format) of A. thaliana, M. truncatula, P. trichocarpa, Zea mays, and Benincasa hispida were downloaded from the NCBI database, and the interspecific collinearity of CSE genes was evaluated by creating a Dual Synteny Plot using the McscanX function in Tbtools.

Plant materials, growth conditions, and pathogen inoculation
Four cucumber varieties, B21-a-2-1-2, B21-a-2-2-2, JinYou 35, and XinTaiMiCi, were used as experimental materials in this study.B21-a-2-1-2 and B21-a-2-2-2 were used to determine gene expression patterns under P. xanthii infection, and JinYou 35 and XinTaiMiCi were used for gene expression pattern analysis under Corynespora cassiicola infection.XinTaiMiCi was also used for gene cloning and genetic transformation.These plant materials were grown in a culture chamber of Shenyang Agricultural University (Shenyang, China) at 25 °C with a photoperiod of 16 h/8 h (day/night).P. xanthii collected from diseased cucumber leaves and C. cassiicola collected from the PDA medium were, respectively, inoculated on 6-week-old cucumber euphylla via a spore-spraying method described in a previous study (Ren 2011).The leaves were harvested at 0, 6, 12, 24, 72, and 144 h post-inoculation (hpi).All harvested samples were quick-frozen in liquid nitrogen and placed in a freezer at − 80 °C until subsequent analyses.Three biological replicates were performed for each sample.

PCR and RT-qPCR validation
Total RNA was isolated from cucumber leaves using a SteadyPure Plant RNA Extraction Kit (Accurate Biotechnology Co., Ltd., Hunan, China), and cDNA was synthesized with an Evo M-MLV Mix Kit with gDNA Clean (Accurate Biotechnology Co., Ltd., Hunan, China) according to the manufacturer's instructions.A pair of primers, pV190-CsCSE-F (CCC GTC AGG ACT TTA CTT AAT GGA TCC TTA CTT GAA ACC AAA CTC CAT ) and pV190-CsCSE5-R (CGA CCT AGA CCT ATA ACT GGA TCC CCA ATA AAC CAT CAT TTT CGTA), were used to clone the CDS-specific fragment of CsCSE5 via PCR using ApexHF HS DNA Polymerase Premix FS (Accurate Biotechnology Co., Ltd., Hunan, China).
The relative expression levels of CsCSE family members were detected on a Bio-Rad CFX96 Real-Time PCR system (Singapore) with Hieff UNICON Universal Blue qPCR SYBR Master Mix (Yeasen Biotechnology Co., Ltd., Shanghai, China).The CsActin gene was used as the internal reference for normalizing gene expression levels.The 2 −∆∆Ct method was used to compute the relative expression profiles of the target genes.The significance of differences in expression levels was estimated using least significant difference (LSD) multiple comparison tests (P ≤ 0.05 or P ≤ 0.01) via IBM SPSS Statistics 26 software.The primers used were designed with Primer 5 software and are shown in Supplemental Table 5.

Cucumber green mottle mosaic virus (CGMMV)-mediated gene silencing
The CGMMV-based pV190 vector has previously been shown to effectively silence genes in cucurbit plants (Liu et al. 2020).In this study, the target sequence of CsCSE5 was cloned from cucumber using gene-specific primers and inserted into the pV190-linearized vector digested with BamHI using homologous recombination to construct pV190-CsCSE5.The pV190 and pV190-CsCSE5 vectors were transformed into Agrobacterium tumefaciens strain GV3101 using the freeze-thaw method, and then monoclonal colonies were cultured in YEP liquid medium containing 50 µg mL −1 kanamycin and 50 µg mL −1 rifampicin overnight at 28 °C.The bacteria were collected by centrifugation and resuspended in infection buffer containing 10 mM MgCl 2 , 10 mM MES, and 100 µM acetosyringone.The OD 600 was adjusted to 0.8-1, and the bacteria were permeated into cotyledons of 2-week-old cucumbers with a 1-mL needleless syringe after they had been maintained in the dark for 3 h at room temperature.The leaves were collected for gene expression analysis at 39 days post-injection; they were then inoculated with P. xanthii and C. cassiicola to further observe and detect fungal biomass.

Coomassie brilliant blue (CBB) staining and disease index (DI) investigation
CBB staining was performed on the leaves of cucumber injected with pV190 and pV190-CsCSE5 at 7 days postinoculation (dpi) to observe the hyphal infection of P. xanthii.First, the leaves of cucumber infected with P. xanthii were immersed in a decolorizing solution containing trichloroacetic acid, chloroform, and absolute ethanol (9:2:6 w/v/v) and treated at 70 °C until the leaves completely faded.The above materials were then soaked in a dyeing solution containing CBB R250, trichloroacetic acid, methanol, and ddH 2 O for 5 min (12:3:2:2 w/w/v/v), rinsed with distilled water, and imaged using a light microscope (Nikon Ts2, Tokyo, Japan).DI was estimated using previously described statistical methods and calculation formulas to quantify the infection of P. xanthii and C. cassiicola on, respectively, inoculated leaves of pV190-transformed and pV190-CsCSE5-transformed cucumbers (Meng et al. 2018).

Identification and characterization of CsCSE genes in cucumber
Searches of cucumber CsCSE genes were conducted in the NCBI database, and the Batch CD-search website was used to further confirm the domain integrity of the candidate genes (Supplementary Table 1 and Supplementary Fig. 1).A total of eight CsCSE genes were obtained from the cucumber genome, which were named CsCSE1 to CsCSE8 according to their locations on the chromosomes (Table 1).We also analyzed the physicochemical properties of the proteins encoded by CsCSE genes.These CsCSE genes encoded proteins varying from 316 (CsCSE5) to 400 (CsCSE6) amino acids with MWs ranging from 36,028.63Da (CsCSE2) to 45,295.65 Da (CsCSE6).The pIs of CsCSE proteins ranged from 5.30 (CsCSE1) to 8.90 (CsCSE6), and most were rich in acidic amino acids.The data presented above suggest that CsCSE proteins may perform various functions in diverse microenvironments.In addition, the GRAVY of all CsCSE proteins was negative, indicating that all members of this family were hydrophilic proteins.With the exception of CsCSE6 and CsCSE7, the instability index of other CsCSE proteins was less than 40 (ranged from 28.26 to 37.28), indicating that they were stable.Signal peptides were not detected in any of the CsCSE family members.These data also suggest that different CsCSE proteins may have the same functions.

Multiple sequence alignment of CsCSE proteins
DNAMAN software was used to conduct a multiple sequence alignment of CsCSE family members.As shown in Fig. 1, the hydrolase domains (GXSXG) and the acyltransferase domains (HX 4 D), two typical conserved domains of CSE, were identified in these CsCSE protein sequences, suggesting that CsCSE may have CSE activity (Gao et al. 2010;Wang et al. 2021a, b;Vijayaraj et al. 2012).With the exception of CsCSE7, which contained one GXSXG domain due to the substitution of the first glycine (G) by serine (S) in the second GXSXG domain, the other CsCSE proteins all contained two GXSXG domains, which reflects the highly conserved nature of this domain.Here, we found that CsCSE3 contains two HX 4 D domains that are not possessed by other CsCSE proteins.In addition, a sequence insertion was detected in CsCSE7.We speculate that the functions of these two proteins may be altered due to the above variations.The CsCSE family has a highly conserved catalytic  triad (S, D, and H) responsible for lipase/esterase activity, which is indicated by the red triangles (Wang et al. 1997).

Gene structure and protein motif analysis of the CsCSE family
Differences in gene structure and the conservation of protein motifs may have important evolutionary implications (Xia et al. 2022).To clarify the structure of CsCSE genes, we analyzed the UTRs, CDSs, and introns of CsCSE genes based on the phylogenetic relationships among members of this family (Fig. 2a and c).The structure of CsCSE genes was variable; the number of UTRs ranged from two to four, the number of CDSs ranged from one to eight, and the number of introns ranged from zero to nine (Fig. 2c).CsCSE1, CsCSE4, and CsCSE6 contained the same number of UTRs (two), CDSs (eight), and introns (seven), yet they belonged to different clades.The same pattern was also observed in CsCSE2 and CsCSE3, both of which contained two UTRs, one CDS, and no introns (Fig. 2a, c).Moreover, the numbers of UTRs, CDSs, and introns of these two genes were the lowest in this family, and the greatest numbers of UTRs, CDSs, and introns were observed in CsCSE8, which contained four UTRs, eight CDSs, and nine introns.In addition to differences in the number of different gene components, the lengths of each gene component varied among CsCSE family members.The longest UTR was 1033 bp (CsCSE7), and the shortest UTR was only 53 bp (CsCSE8).The lengths of CDSs ranged from 3 bp (CsCSE1) to 1002 bp (CsCSE7).
The longest and shortest introns were observed in the CsCSE4 gene (3862 bp and 74 bp, respectively) (Fig. 2c).Differences in the length and number of gene components might underlie the formation of various functional motifs.
To further explore the functional characteristics of CsCSE proteins, the structural motifs of CsCSE proteins were evaluated using the online website MEME, and the results were visualized using TBtools software (Fig. 2b and Supplementary Table 2).A total of 10 conserved motifs were identified in the CsCSE family and named motif 1 to motif 10.No member of the CsCSE family had all 10 conserved motifs, and the number of motifs ranged from six (CsCSE3) to nine (CsCSE2 and CsCSE8).The structure of CsCSE2 and CsCSE3 was similar, but they differed in the number of motifs, which might stem from the fact that these two proteins were placed in different clades (Fig. 2).Motifs 1, 2, 3, and 4 were highly conserved in the CsCSE family; these were considered the characteristic motifs of CsCSE given that they were present in all members of this family.Among them, motif 3 is also considered a classic motif because it contained the GXSXG domain.In addition, some motifs were only present in certain proteins, such as motif 9, which was only identified in CsCSE6 and CsCSE8, and motif 10 was only identified in CsCSE2 and CsCSE3 (Fig. 2b).CsCSE1, CsCSE4, and CsCSE5 contained the same motifs (motifs 1, 2, 3, 4, 5, 6, 7, and 8), suggesting that these three proteins may perform the same function.They were located in the same or similar clades, which reflects the positive relationship between heredity and function, and suggests that the topology of the phylogenetic tree is accurate (Fig. 2a, b).Furthermore, CsCSE2 and CsCSE8 contained the greatest number of conserved motifs and may have additional biological functions; however, further study is needed to confirm this possibility (Fig. 2b).

Cis-acting element analysis of CsCSE promoters
Cis-acting elements in gene promoter regions mediate responses to plant growth and stress by regulating the expression of genes (Walther et al. 2007).To clarify the expression and regulatory characteristics of CsCSE genes and identify their potential biological functions, we used the PlantCARE database to analyze the cis-acting elements in the upstream 2000-bp promoter sequences of CsCSE genes.Various types of typical cis-acting elements were identified, including hormone-responsive elements (salicylic acidresponsive, MeJA-responsive, abscisic acid-responsive, gibberellin-responsive, and auxin-responsive elements), stressresponsive elements (defense and stress-responsive as well as low temperature-responsive elements), growth and development-related elements (light responsiveness, seed-specific regulation, anaerobic induction, anoxic specific inducibility, and zein metabolism regulation), and transcription factorrelated elements (MYB, MYBHv1-binding site, and W-box) (Fig. 3 and Supplementary Table 3).All members of CsCSE gene family contained the elements for light responsiveness.Anaerobic induction and MYB elements were the second most widely distributed in the CsCSE gene family (75% of CsCSE genes), with the exception of CsCSE6 (lack of anaerobic induction), CsCSE8 (lack of MYB), and CsCSE1 (lack of both).A total of 62.5% of CsCSE genes contained salicylic acid-responsive, MeJA-responsive, W-box, and zein metabolism regulatory elements, and 50% of CsCSE genes contained abscisic acid-responsive, gibberellin-responsive, and defense and stress-responsive elements.In addition, low temperature-responsive and MYBHv1-binding site elements were detected in CsCSE4 and CsCSE8, and an anoxic specific inducibility element was also detected in CsCSE4 as well as an auxin-responsive element was also detected in CsCSE8.A seed-specific regulatory element was identified in CsCSE1 (Fig. 3).Overall, the presence of these functional elements suggests that the expression of CsCSE genes might be induced by a variety of hormones and that they are involved in specific growth, development, and signaling pathways as well as stress responses, thereby mediating the resistance of cucumber plants to various environmental stresses during their growth.

Chromosome distribution and collinearity analysis of CsCSE genes
The positions of CsCSE genes in the cucumber genome were visualized using TBtools software.As shown in Supplementary Fig. 2, eight CsCSE genes were unevenly distributed on chromosomes 1, 3, 4, and 5. Chromosome 5 had the greatest number of CsCSE genes: CsCSE5, CsCSE6, CsCSE7, and CsCSE8.Two CsCSE genes (CsCSE1 and CsCSE2) were present on chromosome 1, only one CsCSE gene was present on chromosomes 3 and 4 (CsCSE3 and CsCSE4, respectively), and no CsCSE genes were present on chromosomes 2, 6, and 7. To explore the evolutionary history of CsCSE genes, we detected duplication events in members of this gene family.However, no tandem duplication and fragment duplication events were detected in CsCSE genes, and this reflects the evolutionary conservation of CsCSE genes within species (Supplementary Fig. 2).
Collinear relationships among species provide insights into the evolution of gene families and gene functions (Yao et al. 2022).Collinearity analysis was performed on the genomes of cucumber and five other plants, A. thaliana, barrel medic (M.truncatula), poplar (P.trichocarpa), corn (Z.mays), and wax gourd (B.hispida), to further elucidate the evolutionary history of CsCSE genes (Fig. 4 and Supplementary Table 4).Collinear pairs of CSE genes between cucumber and P. trichocarpa were the most common (seven), followed by A. thaliana (six), B. hispida (six), M. truncatula (five), and Z. mays (three).Moreover, collinear gene pairs of the three cucumber CsCSE members, CsCSE2, CsCSE4, and CsCSE5, were detected in all five species.The above results reveal the evolutionary differentiation of CSE genes between species.Collinear gene pairs of XM_004153438.3(CsCSE1) and XM_004147950.3(CsCSE3) were only detected between cucumber and B. hispida.The formation of these speciesspecific collinear gene pairs might be associated with the evolutionary mechanism of Cucurbitaceae plants, which indicates that these two genes were highly conserved during the evolution of cucumber and B. hispida.In addition, CsCSE genes, with the exception of CsCSE6 and CsCSE8, could be matched with their corresponding CSE genes in B. hispida, which reflects the high homology of CSE genes during the evolution of Cucurbitaceae.

Expression patterns of CsCSE genes in cucumber varieties resistant and susceptible to P. xanthii and C. cassiicola infection
The expression patterns of genes under stress conditions usually reflect their potential functions in stress responses (Zhu et al. 2021).To investigate whether members of the CsCSE family are involved in the defense response of cucumber to pathogens, we evaluated the expression characteristics of eight CsCSE genes in B21-a-2-1-2 (resistant) and B21a-2-2-2 (susceptible) leaves inoculated with P. xanthii as well as JinYou 35 (resistant) and XinTaiMiCi (susceptible) leaves inoculated with C. cassiicola at 0, 6, 12, 24, 72, and 144 hpi (Fig. 5).The expression patterns of these CsCSE genes under infection of the two pathogens were diverse, but some similarities were observed.All eight genes were significantly differentially expressed in resistant and susceptible cucumbers infected with P. xanthii and C. cassiicola at various time points, and changes in expression were observed, which reflects their diverse functional roles in responses to pathogen stress.
Under P. xanthii infection, the expression of CsCSE5 was higher in resistant variety than in susceptible variety.However, the opposite patterns were observed for CsCSE2 and CsCSE6; that is, their expression levels were significantly lower in resistant variety than in susceptible variety.Therefore, we speculated that these two groups of CsCSE genes may be involved in the response of cucumber to attack by P. xanthii but have opposite regulatory roles.The expression peak of CsCSE5 was observed at 144 hpi in both resistant and susceptible variety, and its expression level was relatively low and stable in the early stage of P. xanthii infection, suggesting that CsCSE5 may be involved in regulating cucumber defenses during the late stage of P. xanthii stress.In contrast, the expression peak of CsCSE6 was observed at 0 hpi, and its expression was markedly down-regulated and remained low under P. xanthii infection.The expression of CsCSE1, CsCSE3, and CsCSE8 in susceptible variety also peaked at 144 hpi; unlike CsCSE5 however, the expression levels of these three CsCSE genes remained low in resistant variety.The expression of CsCSE4 fluctuated greatly during P. xanthii infection, and the opposite expression patterns were observed in resistant and susceptible variety.Specifically, the expression of CsCSE4 first decreased and then increased in resistant variety, but opposite was recorded for susceptible variety, which means that this gene might play an important role in all stages of P. xanthii transmission (Fig. 5a).
In addition, the expression patterns of eight CsCSE genes under C. cassiicola infection were also diverse.The expression of CsCSE1, CsCSE4, and CsCSE8 was significantly lower in resistant variety than in susceptible variety, indicating that these three genes might play a negative regulatory role in the resistance of cucumber to C. cassiicola.In contrast, the expression pattern of CsCSE6 was opposite to that of these genes; we speculate that CsCSE6 might positively regulate the resistance of cucumber to C. cassiicola.The expression of all eight genes peaked at 72 hpi in resistant or susceptible variety, and the most pronounced differences in the expression of CsCSE2, CsCSE6, and CsCSE7 in resistant and susceptible variety were observed at this time point, suggesting that this timepoint might be critically important for the resistance of CsCSE genes to C. cassiicola.Aside from CsCSE1, only CsCSE5 was differentially expressed at 144 hpi, indicating that this gene may play a role in regulating the tolerance of cucumber to C. cassiicola during the later stages of infection (Fig. 5b).
In conclusion, all eight CsCSE genes may mediate cucumber-P.xanthii and cucumber-C.cassiicola interactions.The function of the CsCSE5 gene was studied in light of the fact that its differential expression was most pronounced in resistant and susceptible cucumber leaves infected with P. xanthii and C. cassiicola and the fact that it shows highest homology with CsCSE1, which has been shown to play a positive regulatory role in the resistance of cucumber to P. xanthii (Yu et al. 2022) (Figs.2a and 5).

Silencing of CsCSE5 attenuated the resistance of cucumber to P. xanthii and C. cassiicola
To characterize the function of CsCSE5 in mediating the tolerance of cucumber to P. xanthii and C. cassiicola, we generated plants with the CsCSE5 gene knocked down using a CGMMV-based VIGS approach and investigated the effect of CsCSE5 silencing on cucumber disease resistance.As previously mentioned, the pV190 vector modified by CGMMV can be used to induce gene silencing in cucurbit plants (Liu et al. 2020).In this study, a 289-bp specific fragment of the CsCSE5 gene was inserted into the pV190 vector, which yielded the pV190-CsCSE5 recombinant vector (Fig. 6a).The expression of CsCSE5 was significantly reduced in pV190-CsCSE5 plants relative to pV190 plants (Fig. 6b).These results indicated that CsCSE5-silenced cucumber plants had been successfully acquired.
The leaves of these CsCSE5-silenced and control cucumbers were vaccinated with P. xanthii and C. cassiicola to evaluate powdery mildew (PM) and target leaf spot (TLS) disease incidence, respectively.PM and TLS symptoms in both pV190-CsCSE5 and pV190 plants were observed at 7 dpi, but the PM and TLS symptoms of pV190-CsCSE5 cucumbers were particularly severe (Fig. 6c and e).Pictures of P. xanthii hyphae stained with CBB obtained using a microscope indicated that hyphal density was markedly greater in pV190-CsCSE5 cucumbers than in pV190 cucumbers, suggesting that CsCSE5-silencing facilitated the growth of P. xanthii hyphae (Fig. 6d).Moreover, at least 60 pV190-CsCSE5 and pV190 plants infected with P. xanthii and C. cassiicola were used to calculate the DI.The DI of pV190-CsCSE5 plants was higher than that of pV190 plants in both P. xanthii and C. cassiicola (Fig. 6c and e).In summary, the silencing of CsCSE5 in cucumber attenuated resistance to P. xanthii and C. cassiicola, and CsCSE5 was required for the defense of cucumbers against pathogens.

Discussion
Lignin is one of the most important components of plant cell walls; it thus provides a key barrier for plants that prevents the invasion of various pathogens (Cao et al. 2022;Lee et al. 2019).CSE has recently attracted much research attention because it is a key enzyme in the lignin biosynthesis pathway (Vanholme et al. 2013).CSE gene family members that might contribute to plant disease resistance were identified.Although whole-genome sequences of plants can be used to identify and analyze gene families, CSE gene family members have not been extensively studied in cucumber; poplar CSE genes are the most well studied (Wang et al. 2021a, b).Here, we analyzed CsCSE gene family members in cucumber to clarify their molecular functions under pathogen stress and explore the regulatory mechanisms underlying their effects on disease resistance.
We identified a total of eight CsCSE genes in cucumber based on the whole-genome sequence of cucumber, which is less than half the number of PoptrCSE (18) genes in poplar, suggesting that the number of CSE family members may vary among species (Wang et al. 2021a, b).CsCSE genes were nonuniformly scattered on four of seven chromosomes in the cucumber genome (Supplementary Fig. 2).Investigation of the physicochemical properties of CsCSE proteins revealed that most CsCSE family members were acidic and stable proteins as well as all of them were hydrophilic proteins.Furthermore, no signal peptide was found in all CsCSE proteins (Table 1).Multiple sequence alignment data showed that all members of the CsCSE family shared domains (GXSXG and HX 4 D) required for CSE activity.
The structure of genes and conserved motifs of proteins often affect gene function (Swarbreck et al. 2008).Generally, genes in the same clade of a phylogenetic tree have identical or similar gene structures and conserved motifs.CsCSE1 and CsCSE5 were in the same clade and contained identical motifs, which indicates that these two genes may have similar biological functions.CsCSE1, CsCSE4, and CsCSE6 as well as CsCSE2 and CsCSE3 had the same number of gene components, but they did not belong to the same clade.Furthermore, the composition of CsCSE2 and CsCSE3 motifs differed (Fig. 2).These results are inconsistent with our conventional understanding and reflect the diversity and complexity of the CsCSE gene family.In-depth analysis of collinearity between different species can provide insights into the evolution of gene families (Yao et al. 2022).Collinearity between cucumber and five other plant species (A. thaliana, M. truncatula, P. trichocarpa, Z. mays, and B. hispida) showed that six CsCSE genes (excluding CsCSE6 and CsCSE8) showed collinearity with CSE in B. hispida, and fewer homologies were detected in other species.Moreover, collinear relationships between CsCSE1 and CsCSE3 only existed between cucumber and B. hispida.These results reveal the high homology and evolutionary conservation of CSE genes in Cucurbitaceae (Fig. 4 and Supplementary Table 4).Analysis of the cis-acting elements of promoters is significant for predicting the potential biological functions of gene families (Walther et al. 2007).We found that the promoters of CsCSE genes contain a large number of important hormone-responsive elements, stress-responsive elements, growth and development-related elements, and transcription factor-related elements (Fig. 3 and Supplementary Table 3), indicating that CsCSE genes likely play key roles in regulating growth and development as well as stress tolerance.Although CSE genes have been cloned in various plants, such as A. thaliana and M. truncatula, their roles in regulating the stress responses of pathogens have not been widely studied (Vanholme et al. 2013;Ha et al. 2016).We recently showed that the CsCSE1 gene is a key positive regulator of defense against P. xanthii infection in cucumber (Yu et al. 2022).P. xanthii is an obligate trophic fungal pathogen capable of causing PM in cucumber that poses a serious threat to cucumber production (Zhang et al. 2021;Wang et al. 2021a, b).TLS caused by C. cassiicola is another common fungal disease in cucumber culture (Wang et al. 2018).The genetic breeding of tolerant cucumber cultivars is considered the most economically efficient and environmentally friendly strategy for controlling P. xanthii and C. cassiicola, and functional analysis of key disease resistance genes in cucumber is necessary for constructing networks of resistance genes and molecular design breeding (Nie et al. 2015).There is thus a need to identify genes that play a key role in cucumber-P.xanthii and cucumber-C.cassiicola interactions and clarify their potential molecular mechanisms.The expression patterns of genes are closely related to their molecular functions in plants, often crucial for revealing their underlying biological functions.In our study, we assessed the expression patterns of eight CsCSE genes during P. xanthii and C. cassiicola infection to further monitor whether members of the CsCSE family mediate responses to P. xanthii and C. cassiicola infection in cucumber.The expression levels of all eight genes were markedly different in resistant and susceptible variety, and their expression levels intensively changed during P. xanthii and C. cassiicola infection, indicating that these eight CsCSE genes might play indispensable roles in cucumber resistance to P. xanthii and C. cassiicola (Fig. 5).The function of CsCSE5 was characterized based on its expression patterns under pathogen stress and the results of the phylogenetic analysis of CsCSE gene family members.The tolerance of cucumber to P. xanthii and C. cassiicola was significantly attenuated by the silencing of CsCSE5 in cucumber leaves, suggesting that CsCSE5 is a positive regulator of resistance to P. xanthii and C. cassiicola in cucumber, which is consistent with the previously characterized function of CsCSE1 and further confirms the reliability of our data (Fig. 6).This result provides novel insights into the function of CsCSE gene family members in cucumber.
In conclusion, we analyzed the structural features and potential evolutionary relationships of CsCSE gene family members via bioinformatics analyses; we also conducted expression pattern analyses to infer the potential functions of these genes and used CGMMV-based VIGS technology to clarify the function of the candidate gene CsCSE5 under pathogen stress.Our data not only provide information that will aid the cultivation of new disease-resistant cucumber varieties but also have important implications for future studies of CSE-mediated immunity in other plants.

Fig. 1
Fig. 1 Multiple sequence alignment of CsCSE proteins.Two conserved lyase domains (GXSXG) and two typical acyltransferase domains (HX 4 D) are marked by yellow and green boxes, respectively.

Fig. 2
Fig. 2 Phylogenetic relationships, protein motifs, and gene structures of CsCSE gene family members.A The phylogenetic tree of CsCSE proteins was constructed using MEGA7 software.B The motif distribution of CsCSE proteins was determined using MEME software.The 10 motifs marked by their corresponding numbers are displayed

Fig. 3
Fig. 3 Cis-acting elements of CsCSE promotors.Different cis-acting elements are represented by boxes of different colors

Fig. 4
Fig. 4 Collinearity analysis of CSE genes between cucumber and five other representative plant species.The gray lines in the background indicate the collinear blocks within cucumber and other plant genomes, and the red lines highlight the collinear CSE gene pairs (color figure online)

Fig. 6
Fig. 6 Silencing of CsCSE5 attenuated the resistance of XinTaiMiCi cucumber leaves to P. xanthii and C. cassiicola.A Schematic diagram of pV190 and pV190-CsCSE5 vector structures used for the transformation process.B The expression level of CsCSE5 was determined in pV190 and pV190-CsCSE5 cucumbers by RT-qPCR.The expression level of CsCSE5 in pV190 cucumbers was normalized to 1. Data are means ± SD of three biological replicates per treatment.Significant differences were detected by LSD multiple comparison tests

Table 1
Basic information on CsCSE genes