| The ubiquitin-proteasome pathway plays a central role in plant defense responses (including hypersensitive response), the specificity of whose is controlled by E3 ubiquitin ligases which selectively recognize the appropriate protein substrate for breakdown. The aim of the studies reported in this paper was to clone and analyze the ubiquitin ligases-related genes to look into the regulating function of them associated with vertical and horizontal resistance to late blight, or the signal transmitting function of potato prevent late blight infection, and provision of fundamental bases for further study.Two expressed sequence tags (ESTs) (10-A12 and 08-E12) which are similar to tobacco (Nicotiana tabacum) Avr9/Cf-9 rapidly elicited (ACRE) genes were firstly identified from Phytophthora infestans-inoculated leaves of potato (Solanum tuberosum L.) clone 386209.10 which is free from R1-R11 genes. In present study, we cloned two genes standing for these two ESTs, StRFP and StPUB, and elucidated possible functions of them responding to biotic and abiotic stresses.The full-length cDNA of StRFP is of 1354 bp with an open reading frame (ORF) of 789 bp which encodes 262 amino acid residues. The deduced amino acid sequence contains a highly conserved RING-H2 type RING finger domain, a transmembrane region and a GLD signature, implying StPUB is a novel ATL (Arabidopsis toxico para levadura) protein of potato. The Southern blot analysis and mapping indicated that StRFP had one to two copies in potato genome without intron locating on chromosome 3. Transient expression of StRFP:GFP in onion epidermal cells revealed that StPUB localized to the plasma membrane or out of that. RT-PCR assays showed that StRFP was constitutively and differentially expressed in potato plants, including roots, stems, leaves, flowers, shoot tips, stolons and tubers, and significantly induced in potato detached leaves treated with P. infestans, a pathogen causing potato late blight, and plant defense-related signal molecules such as salicylic acid (SA), methyl jasmonate (MeJA) and abscisic acid (ABA), implying StRFP may be involved in potato response to pathogen attack. The results of treating in vitro potato plantlets with environmental stresses showed that StPUB was responsive to NaCl,20% PEG, dehydration,40℃heat and 4℃cold, especially enhanced salt and osmosis tolerance. The function of StRFP in potato resistance against late blight was further investigated by constructing of overexpression and RNA inteference (RNAi) vectors and introducing into potato cv. E-potato 3. There were 27 overexpression and 6 RNAi silencing transgenic lines with 1-3 copies of the gene insertion confirmed by the Southern blot analysis. By challenging the detached leaves with mixture races of P. infestans containing races 1,3,4, and 1.3, all of StRFP-overexpressing transgenic plants displayed a slower disease development than nontansformed control in terms of the lesion growth rate (LGR) and four out of eleven transformants were significant (p<0.05). In contrast, StRFP-silencing lines by RNAi were more susceptible to the pathogen infection and three of four lines showed significantly higher LGR than control. Present results demonstrate that StRFP plays a positive role in the resistance of potato against P. infestans.The full-length of StPUB is of 2483 bp with an ORF of 2175 bp encoding 724 amino acids (GenBank acc. No. EF091878). The StPUB protein contains UND/PUB/ARM repeat domain with 89% of identity to tobacco ACRE276. Furthermore, the intron-exon junction of StPUB gene established by comparison between the cDNA and the genomic sequences from potato cvs.386209.10, E-potato 3 (EP3) and Zhuanxinwu (ZXW) indicated that the sequences maintained high levels (97.7%) of conservation in terms of sequence length and nucleotide composition, and all the gDNAs from the three cultivars seemed to be composed of a single exon without intron. The TAIL-PCR and bioinformatics analysis speculated that StPUB had three promoter sequences and maybe locate on chromosome 2. The DNA gel blot analysis indicated that there are two to three copies of StPUB or closely related genes in the potato genome. The RT-PCR analysis showed a similar expression pattern of StPUB obseverd in the three potato cultivars when detached leaves were infected by P. infestans. The expression of StPUB was upregulated not only in detached leaves by signal molecules of salicylic acid and abscisic acid and wounding, but also in in vitro plantlets by high (40℃) and low (4℃) temperatures and dehydration induced by polyethylene glycol and NaCl. The function of StPUB was further clarified by silencing the gene in cv. E-potato 3 using RNAi-based post-transcriptional gene silencing (PTGS). Thirteen transgenic lines with 1-3 copies of the gene insertion were obtained, and much lower expression of StPUB was observed in transgenic lines than control monitored by RT-PCR. The LGR of StPUB-RN Ai transgenic plants were higher than the control, revealing that silencing of StPUB in potato resulted in a partial loss of plant resistance against late blight. Its function in the salt tolerance was further investigated by subjecting the in vitro plantlets to different concentrations of NaCl stress, transgenic plantlets showed much higher sensitivity to NaCl than control in terms of shoot length, plant fresh weight, rooting ratio and percentage of decayed plantlets, suggesting that StPUB is also involved in response to the salt stress. Present data indicated that StPUB is a gene harboring broad-spectrum responses to both biotic and abiotic stresses in the potato and may play crucial roles in late blight resistance and salt tolerance. |
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