Valsa canker, caused by fungal pathogens in Valsa species, is a fungal disease of apple and pear growing in China and even in Asia. Malectin-like kinases play crucial roles in plant recognition of the pathogen-induced signals and subsequent activation of partially host immune responses. However, the role of MEDOS1 (MDS1), a Malectin-like kinase, in plant immunity has not yet been extensively explored. Here, we found that the expression of the Malus domestica MDS (MdMDS1) gene, a homologous gene of the Catharanthus roseus Receptor-Like Kinase 1-like (CrRLK1L) family member MDS1 in Arabidopsis, could be inhibited by Valsa canker signals. Over-expression of MdMDS1 decreased Valsa canker resistance of apple and pear fruits, as well as 'Duli-G03' (Pyrus betulifolia) suspension cells. In response to Valsa pyri (Vp) signals in suspension cells, the up-regulation of MdMDS1 caused the inhibition of defense-related genes but activated the expression of cell wall-related genes. Among these, the pectin methylesterase gene PbePME1 was robustly induced. Further analysis confirmed that PbePME1, a negative regulator of Valsa canker resistance, was indispensable for MdMDS1's function. Our results enriched the recognization of the functions of CrRLK1L genes in host resistance against necrotrophics. We also provided a theoretical reference for the resistance breeding and comprehensive control of Valsa canker in both apples and pears.

The WRKY transcription factor family plays a crucial role in regulating plant growth and stress responses. However, there are few studies on the regulation of resistance to Valsa canker. In this study, a comprehensive analysis of WRKY genes across 19 plant species was conducted. The potential members of Valsa canker resistance regulation were identified via functional validation. A total of 1641 WRKY genes could be categorized into seven groups. WRKY family members show subfamily- and species-specific expansions. In Rosaceae, Group II-d and II-e were rapidly expanded, which mainly originated based from whole genome duplication (WGD). Cis-element analysis and protein interaction network prediction underscored that most WRKYs respond to stress signals. Based on expressional investigation and Weighted Gene Co-expression Network Analysis (WGCNA), 9 WRKY genes in Pyrus betulaefolia were screened as candidates for Valsa canker resistance regulation. Functional analysis further demonstrated that PbeWRKY16 and PbeWRKY31 regulate the expression of genes involved in salicylic acid (SA) biosynthesis and transport, thereby enhancing resistance to Valsa canker and activating immune responses. Our results provide a foundation for understanding the evolutionary mechanisms of the WRKY gene family and screened potential family members on Valsa canker resistance regulation.

Abstract Receptor-like proteins (RLPs) are crucial pattern-recognition receptors on the surface of plant cells, which are involved in almost all processes of the plant life cycle. Recently, the evolution of high-throughput sequencing technology has strengthened the appraisal and identification of increasing numbers of RLPs and has primarily improved our understanding of the roles of RLPs in various biological processes. Here, we review the classification and evolutionary characteristics of RLPs and their regulatory roles in pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). In particular, we summarize the ligands recognized by RLPs, their co-receptors, and downstream signalling cascades mediated by RLPs. To summarize, this review offers beneficial guidance for researchers in at-a-glance comprehension of the function of RLPs. It also puts forward the prospect of mining broad-spectrum candidate genes in light of the research on the disease resistance mechanism of RLPs and current challenges in disease resistance breeding.

Wall-associated kinases (WAKs) have been determined to recognize pathogenic signals and initiate plant immune responses. However, the roles of the family members in host resistance against Valsa canker, a serious fungal disease of apples and pears, are largely unknown. Here, we identified MbWAK1 in Malus baccata, a resistant germplasm differentially expressed during infection by Valsa mali (Vm). Over-expression of MbWAK1 enhanced the Valsa canker resistance of apple and pear fruits and 'Duli-G03' (Pyrus betulifolia) suspension cells. A large number of phloem, cell wall, and lipid metabolic process-related genes were differentially expressed in overexpressed suspension cell lines in response to Valsa pyri (Vp) signals. Among these, the expression of xyloglucan endotransglucosylase/hydrolase (XTH) gene PbeXTH1 and sieve element occlusion B-like (SEOB) gene PbeSEOB1 were significantly inhibited. Transient expression of PbeXTH1 or PbeSEOB1 compromised the expressional induction of MbWAK1 and the resistance contributed by MbWAK1. In addition, PbeXTH1 and PbeSEOB1 suppressed the immune response induced by MbWAK1. Our results enriched the molecular mechanisms for MbWAK1 against Valsa canker and resistant breeding.

The effect and potential mechanism of receptor like kinase gene MdLYK1(MD09G1111800)on the resistance to Valsa canker were investigated by bioinformatics analysis,functional verification and expression analysis.The results showed that MdLYK1 was homologous to Arabidopsis receptor kinase gene AtLYK1,with 68.1％amino acid sequence similarity.The transient expression of this gene in'Yanfu 6'fruit significantly improved the fruit Valsa canker resistance.Three overexpressed cell lines were obtained by introducing them into the suspension cells of Pyrus betulifolia Bunge.After inoculation with Valsa pyri,the growth rate of colonies on all overexpressing cell lines was significantly lower than that of wild-type cells.All overexpression cell lines were significantly less sensitive to Valsa pyri metabolites compared to the wild type.In addition,MdLYK1 overexpression significantly enhanced the up-regulated expression of key genes related to signaling such as pathogen-associated molecular patterns triggered immunity,reactive oxygen species,jasmonic acid and other signal-related key genes were up-regulated in the suspension cells of Pyrus betulifolia Bunge.In conclusion,MdLYK1 positively regulates the resistance to apple and pear Valsa canker,and pathogen-associated molecular pattern-triggered immunity,reactive oxygen species and jasmonic acid signals are involved in the regulation of resistance.

Valsa canker, caused by the necrotrophic fungus Valsa species, is one of the most destructive diseases that threatens the development of apple and pear production. The discovery of resistance genes is helpful for resistance breeding and for carrying out control measures. In this study, we identified a LysM-Containing Receptor Protein 1 (PbeLYP1) in the Valsa-resistant rootstock Pyrus betulifolia. Expression assays revealed that PbeLYP1 was induced by both Valsa pyri (Vp) and Vp metabolism (VpM). The transient expression on pear fruit and stably transformed suspension cells confirmed the positive role of PbeLYP1 in resistance to Valsa canker. Key genes related to reactive oxygen species (ROS) and jasmonic acid (JA) signaling were significantly induced in PbeLYP1-overexpressing cell lines. In conclusion, PbeLYP1 positively regulates Valsa canker resistance,which is associated with the induction of ROS and JA signaling.

Valsa canker is a fungal disease of apple and pear growing areas in China even Asia. Malectin like kinases take crucial roles in plant recognition of the pathogen-induced signals and subsequent activation of partially host immune responses. However, the role of MEDOS1 (MDS1) in plant immunity has not yet been extensively explored. In the Malus domestic genome, we found that the MdMDS1 , a homologous gene of MDS1 in Arabidopsis , responded to Valsa mali ( Vm ) and Valsa pyri ( Vp ). Over-expression of MdMDS1 decreased Valsa canker resistance of Pyrus betulifolia ‘Duli-G03’ suspension cells, and apple and pear fruits. During suspension cells in response to Vp signals, up-regulation of MdMDS1 inhibited expression of defense-related genes but activated cell wall related genes. Among these, pectin methylesterase gene PbePME1 was robustly induced. Further analysis confirmed that PbePME1 , a negative regulator of Valsa canker resistance, was indispensable for Md MDS1’s function by interaction. Our results provide a theoretical reference for the resistant breeding and comprehensive control of Valsa canker of both apple and pear.

在植物响应逆境过程中,半胱氨酸富集类受体激酶(Cysteine-rich receptor like kinase,CRK)起重要的调控作用.本研究中以结构域Stress-antifung (Pfam:PF01657)和Pkinase (Pfam:PF00069)的保守序列为种子序列,在全基因组范围鉴定了梨(Pryus spp.)CRK家族成员.此外,对其蛋白理化性质、进化特征、基因在染色体上的位置、顺式作用元件(cis-acting regulatory element,cis-element)和表达模式进行了分析.共获得32个CRK家族成员,其氨基酸数、分子量和等电点分别介于440～1 217、48.90～137.02kD和5.31～8.59,主要位于质膜.根据进化分析,将来自梨、拟南芥、苹果、番茄和水稻的156个CRK分为6个亚组,梨CRK主要分布于亚组Ⅳ,V和Ⅵ.梨CRK中存在5个串联重复的基因簇,共包含了20个成员.此外,该基因家族所有成员的启动子区域都存在多个响应激素和逆境信号的cis-element.接种腐烂病菌Valsa pyri(Vp)后,杜梨(Pyrus betulifolia,抗病)和‘早酥’梨(Pyrus bretschneideri,感病)中分别发现8个和10个CRK发生了差异表达,6个基因在两种资源中都发生了差异表达.差异基因中,Pbr001477.1和Pbr000205.4在抗、感病资源中都显著上调,而其他基因的表达量在两种资源中呈现不同的变化趋势.

Rosaceae is an economically important plant family that can be affected by a multitude of pathogenic microbes, some of which can cause dramatic losses in production. As a type of pattern-recognition receptor, receptor-like proteins (RLPs) are considered vital regulators of plant immunity. Based on genome-wide identification, bioinformatic analysis, and functional determination, we investigated the evolutionary characteristics of RLPs, and specifically those that regulate Valsa canker, a devastating fungal disease affecting apple and pear production. A total of 3028 RLPs from the genomes of 19 species, including nine Rosaceae, were divided into 24 subfamilies. Five subfamilies and seven co-expression modules were found to be involved in the responses to Valsa canker signals of the resistant pear rootstock Pyrus betulifolia 'Duli-G03'. Fourteen RLPs were subsequently screened as candidate genes for regulation of resistance. Among these, PbeRP23 (Chr13.g24394) and PbeRP27 (Chr16.g31400) were identified as key resistance genes that rapidly enhance the resistance of 'Duli-G03' and strongly initiate immune responses, and hence they have potential for further functional exploration and breeding applications for resistance to Valsa canker. In addition, as a consequence of this work we have established optimal methods for the classification and screening of disease-resistant RLPs.

Rosaceae is one of the major families in the plant kingdom with important economic value. However, many of them are attacked by Valsa canker, resulting in serious loss of production and profits. Nucleotide-binding leucine-rich repeats (NLRs) play a key role in the plant immune response as the largest class of resistance genes. Currently, we performed a genome-wide identification of NLR genes in Rosaceae and revealed some NLR genes in response to Valsa canker using multispecies bioinformatics including co-expression network analysis and RNASeq data. A total of 3718 NLR genes were identified from genomes of 19 plant species (include 9 Rosaceae plants) and classified them into 15 clades. The NLRs display species- and group-specific expansions that are derived from both the whole genome duplication and the tandem duplication. Additionally, the expression of some NLR members was low under normal growth conditions in various plant tissues, while significantly enhanced after the infection of Valsa canker. Furthermore, co-expression network analysis shows that the 13 NLR members were distributed in key nodes of differentially expressed genes which could be considered as promosing key regulators for the resistance of Valsa canker. Therefore, our findings provide a reference for the evolution of NLR genes in Rosaceae and the key regulators of Valsa canker resistance.

Based on bioinformatics analysis,functional verification,and expression measurement,the role of the receptor-like protein MbRLP7 in Valsa pyri resistance,and its potential regulatory mechanisms were explored.The results showed that MbRLP7 had the highest sequence similarity with AtRLP6 and AtRLP7,with 43.0％and 42.8％,respectively.There are several cis-elements in the promoter region of MbRLP7 in response to stress-related signals and Valsa pyri signals.The MbRLP7 was transformed into suspension cells of Pyrus betulifolia Bunge,and three overexpressed cell lines were obtained.After inoculation with the pathogen Valsapyri(Vp),the growth rate of mycelia on the overexpressing cells was significantly higher than that on the wild-type cells.After Valsapyri metabolites treatment,the cell activity of all overexpressed cell lines was significantly lower than that of wild-type.In addition,the overexpression of gene MbRLP7 enhanced the induction of Valsa pyri metabolites on PAMP-triggered immunity(PTI),reactive oxygen species related genes and pathogenesis-related protein PR1.In conclusion,MbRLP7 negatively regulates the resistance of Pyrus betulifolia Bunge to Valsa pyri,and PTI,reactive oxygen species and PR1 are involved in this regulatory process.

L-type lectin receptor-like kinases (L-LecRKs) act as sensors of extracellular signals and as initiators for plant immune responses; however, the function of LecRK-S.4 in plant immunity has not yet been extensively investigated. In the present study we found that MdLecRK-S.4.3 in apple (Malus domestica), a homologous gene of LecRK-S.4, was differentially expressed during infection by Valsa mali and Valsa pyri. Overexpression of MdLecRK-S.4.3 facilitated the induction of immune responses and enhanced the resistance to Valsa canker of fruits of apple and pear (Pyrus betulifolia), and of suspension cells of pear 'Duli-G03'. The expression of PbePUB36, a RLCK XI sub-family member, was significantly repressed in the MdLecRK-S.4.3-overexpressing cell lines. Overexpression of PbePUB36 interfered with the resistance to Valsa canker and the immune response caused by up-regulation of MdLecRK-S.4.3. In addition, we found that MdLecRK-S.4.3 interacted with BAK1 and/or PbePUB36 in vivo. Thus, whilst MdLecRK-S.4.3 activated various immune responses and positively regulated Valsa canker resistance, this could be largely compromised by PbePUB36. MdLecRK-S.4.3 interacted with PbePUB36 and/or MdBAK1 to mediate the immune responses. Our finding provides a basis for further examination of the molecular mechanisms underlying resistance to Valsa canker, and can contribute to resistance breeding.

Valsa canker is a fungal disease of apple and pear growing areas in China even Asia. Malectin like kinases take crucial roles in plant recognition of the pathogen-induced signals and subsequent activation of partially host immune responses. However, the role of MEDOS1 (MDS1) in plant immunity has not yet been extensively explored. In the Malus domestic genome, we found that the MdMDS1, a homologous gene of MDS1 in Arabidopsis, responded to Valsa mali ( Vm) and Valsa pyri ( Vp). Over-expression of MdMDS1 decreased Valsa canker resistance of Pyrus betulifolia ‘Duli-G03’ suspension cells, and apple and pear fruits. During suspension cells in response to Vp signals, up-regulation of MdMDS1 inhibited expression of defense-related genes but activated cell wall related genes. Among these, pectin methylesterase gene PbePME1 was robustly induced. Further analysis confirmed that PbePME1, a negative regulator of Valsa canker resistance, was indispensable for MdMDS1’s function by interaction. Our results provide a theoretical reference for the resistant breeding and comprehensive control of Valsa canker of both apple and pear.

Pear, one of the staple fruit crops in the world, is under threat from Valsa pyri (Vp) infection. Investigations of the molecular mechanisms underlying pear resistance against Vp infection are largely limited. Using RNA sequencing, we investigated the global gene expression profiles of suspension cells from the resistant rootstock 'Duli-03 ' (Pyrus betulifolia Bunge) and a susceptible cultivar 'Yuluxiang' (P. bretschneideri Rehd.) in response to Vp infection. Differentially expressed genes (DEGs) mainly involved in "Cell wall biosynthesis", "Signal transduction", "Plant-pathogen interaction", "Plant hormone signal transduction", etc., were studied. Further investigations revealed that genes associated with cell wall, Ca2+, hypersensitive response (HR), defense response, and salicylic acid (SA) participated in pear responses to Vp signals. Functional analysis revealed that defenserelated genes PbeKCS1/10-1 and PbeMPK4-1 positively regulated Vp resistance in pear fruit, whereas four TGACG motif binding factor (TGA) genes played a negative regulatory role.

The necrotrophic pathogen Valsa mali (Vm) resulting Valsa canker of apple is considered one of the most destructing fungal diseases. To study the resistance mechanism, we investigated gene expression profiles of suspension cells from resistant varieties ‘Dongbeishanjingzi’ (DS, Malus baccata) and susceptible varieties ‘Gala’ (GL, Malus × domestica) in response to Vm metabolism (VmM) using RNA sequencing (RNA-seq). Functional enrichment showed that differentially expressed genes (DEGs) were widely involved in multiple metabolisms or signals, such as “Lipid metabolic process”, “plant hormone signal transduction” and “plant-pathogen interaction”. Further expressional patterns exhibited that induction of genes related to ‘‘xyloglucan biosynthetic process’’ and ‘‘cell wall biogenesis’’ was beneficial for cell wall integrity and tolerance of DS cells. In brassinosteroid signaling, we identified that TCH4 gene MbTCH4-1 positively regulated Vm resistance of ‘Fuji’ fruit, but BSK gene MdBSK1 negatively regulated the resistance. In contrast, cell death associated with hypersensitive response caused by up-regulation of CNGCs and CDPK genes is an important cause of weakened tolerance in GL cells. Our results provide a new insight direction for the molecular mechanism of apple against Valsa canker.

Pear production was seriously threatened by Valsa canker, a notorious disease caused by the necrotrophic fungus Valsa pyri (Vp), but the molecular mechanism is elusive. Currently, the dynamic gene expression profiles of main susceptible variety 'Zaosu' (Pyrus bretschneideri Rehd.) in response to Vp were investigated using RNA sequencing (RNA-seq). In total, 286, 1912, and 6321 differentially expressed genes (DEGs) were detected from the infected phloem tissues at 1, 2, and 4 days after infection, respectively. Functional terms "Stress biotic", "Signaling. receptor kinase", and "Plant hormone signal transduction" were significantly enriched. DEGs associated with Ethylene, Chitinase, Glycosyl hydrolase, and receptor-like kinase (RLK) subfamilies LysM and WAK_LRK10L-1 were mainly up-regulated and contributed to induction of the immune responses in 'Zaosu'. On the contrary, DEGs involved in auxin and brassinosteroid signals, receptor-like protein, nucleotide-binding leucine-rich repeat (NB-LRR), and RLK subfamilies CrRLK1L-1, LRR-III, LRR-VII, and LRR-XII are mainly down-regulated, which are the reasons why 'Zaosu' fails to prevent Vp invasion. Our results highlight new insights into pear responses against Vp and perhaps other necrotrophs.

该研究结合蔷薇科数据库,经多序列比对、表达分析和验证,筛选了苹果和梨抗病反应Marker基因及其检测引物,并以腐烂病抗性资源杜梨和东北山荆子悬浮细胞为材料,经20％腐烂病菌(Vp)代谢物处理,采用实时荧光定量PCR(RT-qPCR)分析Marker基因对腐烂病信号响应的表达模式,为苹果、梨抗病反应的快速检测和杜梨抗腐烂病机理的系统研究奠定基础.结果显示:(1)经检索比对共筛选出水杨酸(SA)、茉莉酸(JA)、乙烯(ET)、系统获得型抗性(SAR)反应、病原相关分子模式激发的免疫反应(PTI)、植保素和防御相关等信号的15个Marker基因及其对应引物.(2)各处理cDNA浓度采用100 ng·μL-1时Cq值均介于18～25,且PCR扩增效率高,表明所选的15个Marker基因的引物均可准确反映抗性反应的激活状况,可作为各自信号通路的Marker基因.(3)与对照相比,Vp代谢物处理后杜梨悬浮细胞中ROS的积累随处理时间的延长呈逐渐显著上升的趋势,并于处理6 h时,ROS信号值达到对照的3.2倍,表明Vp代谢物会激活体内的免疫反应(PTI),进而导致植物体内ROS的爆发.(4)RT-qPCR验证分析显示,Vp代谢物处理后,杜梨悬浮细胞中SA、JA、PTI、SAR、R基因、植保素、防御相关等信号通路的Marker基因都呈上调表达趋势,其中,SA、JA和SAR通路的Marker基因ChiV(Chitinase class V)、LOX1(Lipoxygenase 1)和PR4(Pathogenesis-Related 4)及防御相关基因PAL1(Phe Ammonia Lyase 1)的表达上调最为明显,最高分别可上升至对照的1718、691、6369和1072倍,表明杜梨受到Vp代谢物胁迫时,主要激活了植物体内的SA、JA、SAR和防御相关信号通路的基因,进而能够抵御病原的进一步入侵.研究发现,SA、JA、SAR和防御反应等信号参与了杜梨对腐烂病胁迫的响应,进而提高其抗病性.

Valsa canker, caused by Valsa pyri (Vp), is a necrotrophic fungal disease that seriously damaged the pear industry in the world. Investigations on resistant mechanisms are helpful for resistance breeding and effective control methods. The current study obtained suspension cells of a resistant rootstock 'Duli-G03' (Pyrus betulifolia bunge) and a susceptible species 'Yuluxiang' (Pyrus bretschneideri Rehd.). Furthermore, the study explored the culture methods of callus and suspension cells of both varieties. Murashige and Skoog (MS) medium supplemented with 0.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/L 6-benzylaminopurine (6-BA) was suitable for callus induction and suspension cell proliferation of 'Yuluxiang', while Nitsch & Nitsch Medium (NN69) with 5 mg/L 6-BA and 0.4 mg/L 1-naphthlcetic acid (NAA) was suitable for 'Duli-G03'. After suspension cells treated with Vp metabolites (VpM), differentially expressed genes (DEGs) were mainly involved in "signal transduction", "plant-pathogen interaction", "plant hormone signal transduction". Among these, disease resistance protein- and tobacco mosaic virus (TMV) resistance protein-related genes, such as GWHGAAYT037865, GWHGAAYT031455, GWHGAAYT037851, were only promptly induced in 'Duli-G03'. On the contrary, DEGs related to Hypersensitive response (HR) (gene4043), ethylene (ET) (gene26613) and brassinosteroid (BR) (gene33208) were aroused only in 'Yuluxiang'. The results supplied important implications for understanding the molecular mechanisms of pear resistance to Valsa canker. Some of the screened genes can be further investigated for disease resistance effects and pave the ground for the mining of disease resistance genes.