The Molecular Mechanism Of Powdery Mildew Resistance In Chestnut Rose (Rosa Roxburghii Tratt)

Chestnut rose (Rosa roxburghii Tratt) has brought about great interest due to thehighest content of vitamin C and supcroxide disomutase (SOD) activity, together withattractive senescence-retarding and cancer-preventing effects. However, powdery mildewdisease is common in the production area, especially when a great number of chestnutrose are cultivated. Injury due to powdery mildews includes stunting and distortion ofleaves, buds, growing tips, and fruit, the symporoms become more serious in the highhumidity area with hot weather. Powdery mildew resistance breeding becomes one of themost important goals for fruit tree breeders, and the prerequistc is genetic knowledge ofdisease resistance mechanisms. The aim of this study is to understand the mechanisminvolved in powdery mildew resistance in chestnut rose.1. From the pathogeneic aspect, we studied the annual life-cycle for the powderymildew fungi and observed the shapes of fungi in different stages, includingconidiophores, conidium, ascospore, hyphae and ascocarps. Moreover, H₂O₂ wereobserved accumulating near around the hyphae attacking sites, and the callose wasdetected by fluorescence analysis. For enzymes, chitinase and giucanase weresignificantly responded to the innoculation of powdery mildew.2. Using homology-based method, resistance gene aanalogues (RGAs) were clonedfrom chestnut rose genome. Sequence polymorphisms, phylogenetic evolution, geneticmapping, relationship with resistance gene loci, and flanking genes were analysed amongthese RGA genes. In further, the RGA genes from chestnut rose were comparativelyanalysed with RGA genes from fruit crops in the Rosaceae. Ninty-six resistance GeneAanalogues (RGAs) were cloned and characterized, of which 34 were derived fromresistant parent—Guinong No. 6, 30 from susceptible parent—Guinong No. 5, 32 from F₁progeny. Comparison revealed that the nucleotide similarity between the resistant andsusceptible parent is averaged at 54%, higher than that within resistant parent.Phylogenetic analysis separated these 96 genes into two groups; one group showed highhomology with nonTIR resistance genes, the other is highly homologous to TIRresistance gene. Genetic mapping divided these 96 genes into 3 linkage groups: thebiggest contained 23 genes, designated as CR1; the second contained 12 genes, all werefrom susceptible parent; the last group CR3 contained 6 genes. A resistance QTL wsadetected in the third group using F₁ population and the disease index data, the nearestRGA gene to this QTL is RGA22. When compare the positions of genetic map withphylogenetic tree, the RGA gene from a phylogenetic clade tended to cluster in geneticmap, which was caused by tandem duplication and diversification. The other case is RGAgenes from different clades clustered together in the genetic map and form heterogeneouscluster, this might caused by ecotopic recombination. From the parents to F₁ progeny,RGA gene's fragment deletion was observed, which may related with meiotic instability.Using TAIL-PCR strategy, we isolated a retrotransposon-like gene at the flank of RGAgene, Southern analysis revealed that the copy number of retrotransposon-like gene isrelative large and exist great differentc among different materials. Based on the pulbished rosaceae RGA genes from chestnut rose, apple, peach, pear, strawberry, apricot and plum,we comparatively analysed on genus and species level. The synteny of RGA genebetween different genus were discussed, and the 125 H site was detected to be underpositive selection by evlutionary prediction analysis.3. In chestnut rose genome, defense-related genes exist in the form of gene family.Among the members, single nucleotide polymorphisms (SNPs) are more prevalent thanother sequence polymorphisms. Several members are obviously responded to the powderymildew attack. From the resistant cultivar——Guinong No. 6, 30 defense-related genes(DR genes) were cloned, including 9 PTO-like kinase genes, 21 pathogenesis-relatedgenes (12 PR2 genes and 9 PR5 genes). The polymorphism of gene family was mainlycomposed by single nucleotide polymorphisms (SNPs). The average frequency for PR5gene was one SNP 59 bp, every 64 bp for PR2 genes. Based on the SNPs, we developedSNAP markers, 23 primer pairs were designed, and 17 markers were finally mapped in F₁population. Reverse Northern revealed that several PR2 genes expression level wasenhanced after innoculation, one PR5 gene was significantly enchanced.4. We proposed a new mechanism for disease resistance—photorespiration.Suppression subtraction hybridization (SSH) library which enriched powdery mildewresponded genes was constructed, and reverse northern technology was used to screen300 clones from the library. Sequencing revealed that the most common genes appearedin the library is photorespiratory-related genes, such as ribulose bisphosphate carboxylase(Rubisco), Rubisco activase, glyoxylate aminotransferase, glutamine synthetase,glyceraldehyde dehydrogenase, ferredoxin, transport protein, etc. Other genes were sameto previously reported genes, such as PR10,P450,STK-like kinase gene, Cf-9-1ike, LRRreceptor-like genes, transcription factor NAC gene, and the transposon genes. Real-TimePCR were taken to verify the three most important photorespiratory genes, whichrevealed that Rubisco(named as ChrRBCs), Rubisco activase (ChrRBCA), glyoxylateaminotransferase (ChrSGT) expression levels were significantly enhanced afterinnoculation. The enzymatic activities of these three photorespiratory genes were alsoverified to be enhanced significantly after innoculation by spectrophotometric analysis.Genetic mapping showed that the three photorespiratory genes exist in the chesnut rosegenome in a form of cluste.We further obtained the full length cDNA sequence of thesethree genes by RACE strategy. Moreover, we compared the DNA regions of these genesamong the genomes of chestnut rose different genotypes and rosaceae fruit crops.Interestingly, the sequence of ChrRBCs in Guinong No.6, which is highly resistant topowdery mildew, is identical to that in Wuzi Cili, a genotype immune to powdery mildew.Altogether, photorespiration may be another physiological process which is involved indefense against pathogen attack in plants.