Abstract :【Objective】 Valsa canker, caused by necrotrophic fungi in Valsa species, is a destructive disease attacking apple and pear production in China and even in East Asia. It is difficult to control the occurrence of this disease through traditional practices due to the extension of mycelium into the xylem. For a long time, resistant breeding has been a widely approved approach but largely limited by delayed progress. At present, it is urgent to identify key genes regulating resistance and related molecular mechanisms. Plants evolved innate immune systems, including horizontal and vertical resistance. Plant cell membrane-localized pattern recognition receptors (PRRs), responsible for recognizing the signals from pathogens and initiating downstream immune responses, are crucial for plant resistance against the infection of various pathogens. Receptor-like kinases (RLKs) are one of the most important PRRs and play vital roles in plant immunity. CRINKLY4 (CR4), a subfamily of RLK, has beenconfirmed as a key regulator for plant resistance. Shanjingzi (Malus baccata) is widely used as the rootstock of apples in China due to its strong tolerance against both biotic and abiotic stresses. In the current investigation, we identified a CR4 member, MbCCR4, which positively regulates Valsa canker resistance. We further analyzed potential signals involved in MbCCR4-regulated immune response. 【Methods】 The domain composition and cis-elements in the promoter region of MbCCR4 were detected by using the online software SMART and Plant CARE, respectively. The phylogenetic relationships between MbCCR4 and the homologous gene in other plant species were analyzed using multiple sequence alignment and phylogenetic tree construction. In order to identify potential roles in Valsa canker resistance, the expression patterns of MbCCR4 were assayed during the Shanjingzi suspension cells responded to Valsa canker signals. The CDS of MbCCR4 was cloned into the expression vector pFGC5941. By using the Freeze-thaw method, the recombined plasmids were transformed into Agrobacterium tumefaciens GV3101. To analyze the roles of MbCCR4 on Valsa canker resistance, the A. tumefaciens carrying empty vector and recombine plasmids were transiently expressed in fruits of ‘Yanfu-3’ (Malus × domestica) and ‘Huangguan’ (Pyrus bretschneideri) and overexpressed into suspension cells of Duli-G-03 (P. betulifolia). The resistance was evaluated by the test of fruits and suspension cells challenged to both pathogens (Valsa mali or V. pyri) and V. mali or V. pyri metabolisms (VmM or VpM). The immunity-related genes, including the genes involved in the signals of pattern-triggered immunity (PTI), reactive oxygen species (ROS), salicylic acid (SA) and jasmonic acid (JA), were detected in wild type and over-expressed cells during VpM exposure. 【Results】 Sequence BLAST assays reveal that the target gene is homologous with CCR4 (AT5G47850.1) in Arabidopsis and is then named MbCCR4. Based on the phylogenetic tree of 30 homologous from 17 plant species, MbCCR4 showed the highest homologousity with MD08G1217500 in Apple. MbCCR4 is a typical member of the CR4 family, which contains an N-terminal signal peptide, a regulator of chromosome condensation 1 (RCC1) domain and a C-terminal kinase domain. Cis-acting regulatory elements (cis-elements) prediction exhibits that MbCCR4 possesses the cis-elements respond to multiple signals such as methyl jasmonate (MeJA), abscisic acid (ABA), and stresses. During ‘DongbeiShanjingzi’ (M. baccata) suspension cells responding to signals from Vm, the expression of MbCCR4 was robustly induced at 1 h. Transient expression showed that up-regulation of MbCCR4 significantly enhanced the Valsa canker resistance of both apple and pear fruits compared to the control (empty vector). Expressional assays exhibit that the target gene was successfully expressed in both apple and pear fruits. Furthermore, MbCCR4 was successfully over-expressed in ‘Duli-G03’ suspension cells. For the three overexpression cell lines MbCCR4-OE1, MbCCR4-OE2 and MbCCR4-OE6, the expression of MbCCR4 were increased to 11.1, 6.8 and 5.8 folds of that in wild type cells. Consistent with transient expression, over-expression of MbCCR4 significantly promoted the tolerance of suspension cells against both Vp and VpM. Compared to the wild type, over-expression of MbCCR4 significantly inhibits the growth ratio of Vp mycelium on suspension cells. At 1 h, 3 h and 6 h of VpM (20%) exposure, the over-expressed cells display higher viability than that of wild type cells. We further detected the expression of marker genes that relate to multiple immune signals in wild type and over-expressed cell lines that respond to VpM signals. The results display that the expression of marker genes associated with multiple signals, such as PTI, ROS, SA and JA, is obviously induced in overexpressed cells. Among these, the expression of SA related gene pathogenesis related protein 1 (PR1) and 4 (PR4) were robustly up-regulated at all time points, indicating the involvement of SA signals in MbCCR4 induced immunity. 【Conclusion】 The above results indicate that MbCCR4 is a key gene that contributes to the Valsa canker resistance of both apples and pears. Moreover, SA is involved in MbCCR4 mediated immune responses. These investigations supplied a valuable gene for further resistant breeding and a theoretical basis for carrying out comprehensive measures to effectively control the occurrence of Valsa canker in apples and pears.
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