Abstract
BackgroundDue to the variation and mutation of the races of Pseudoperonospora cubensis, downy mildew has in recent years become the most devastating leaf disease of cucumber worldwide. Novel resistance to downy mildew has been identified in the wild Cucumis species, C. hystrix Chakr. After the successful hybridization between C. hystrix and cultivated cucumber (C. sativus L.), an introgression line (IL5211S) was identified as highly resistant to downy mildew. Nucleotide-binding site and leucine-rich repeat (NBS-LRR) genes are the largest class of disease resistance genes cloned from plant with highly conserved domains, which can be used to facilitate the isolation of candidate genes associated with downy mildew resistance in IL5211S.ResultsDegenerate primers that were designed based on the conserved motifs in the NBS domain of resistance (R) proteins were used to isolate NBS-type sequences from IL5211S. A total of 28 sequences were identified and named as cucumber (C. sativus = CS) resistance gene analogs as CSRGAs. Polygenetic analyses separated these sequences into four different classes. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that these CSRGAs expressed at different levels in leaves, roots, and stems. In addition, introgression from C. hystrix induced expression of the partial CSRGAs in cultivated cucumber, especially CSRGA23, increased four-fold when compared to the backcross parent CC3. Furthermore, the expression of CSRGA23 under P. cubensis infection and abiotic stresses was also analyzed at different time points. Results showed that the P. cubensis treatment and four tested abiotic stimuli, MeJA, SA, ABA, and H2O2, triggered a significant induction of CSRGA23 within 72 h of inoculation. The results indicate that CSRGA23 may play a critical role in protecting cucumber against P. cubensis through a signaling the pathway triggered by these molecules.ConclusionsFour classes of NBS-type RGAs were successfully isolated from IL5211S, and the possible involvement of CSRGA23 in the active defense response to P. cubensis was demonstrated. These results will contribute to develop analog-based markers related to downy mildew resistance gene and elucidate the molecular mechanisms causing resistance in IL5211S in the future.
Highlights
Due to the variation and mutation of the races of Pseudoperonospora cubensis, downy mildew has in recent years become the most devastating leaf disease of cucumber worldwide
Amplification and cloning of CSRGAs from IL5211S Using a pair of degenerate primers, primer-R and primer-F, from previously published literature [31], a band of the predicted size (~500 bp) was observed by PCR amplification
According to the principle that the W residue is found in non-toll and interleukin-1 receptor (TIR) proteins, and the D residue is only in TIR-containing proteins, 24 out of 28 (82%) of the CSRGAs examined in this work belonged to the TIR-nucleotide binding site (NBS)-leucinerich repeats (LRR) type
Summary
Due to the variation and mutation of the races of Pseudoperonospora cubensis, downy mildew has in recent years become the most devastating leaf disease of cucumber worldwide. Nucleotide-binding site and leucine-rich repeat (NBS-LRR) genes are the largest class of disease resistance genes cloned from plant with highly conserved domains, which can be used to facilitate the isolation of candidate genes associated with downy mildew resistance in IL5211S. Some are specific to the non-TIR or the TIR-NBS-LRR subfamily, such as RNBS-A-TIR and RNBS-D-TIR in the TIR subclass, and RNBS-A-nonTIR and RNBS-D-nonTIR in the nonTIR subclass [8,9] Other conserved motifs such as P-loop (kinase-1a), kinase-2, kinase-3a, and GLPL are widely present in both classes. Conserved motifs in such resistance genes in different plants offer a way to isolate RGAs related to other resistance genes. Degenerate RCR could be a promising approach that may facilitate the isolation of resistance genes
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