Molecular Mechanism Of Wheat And Puccinia Striiformis F. Sp. Tritici Interactions And Functional Characterization Of Resistance-Related Genes

Stripe rust, caused by Puccinia striiformis Westend. f. sp. tritici Eriks. (Pst), is a devastating disease in wheat worldwide. The use of resistance cultivars offers the most economic and environmental friendly way to control the disease. In China, we have made great achievements on the control of wheat stripe rust through research on epidemiology and control of the disease. Systemic research on the mechanisms between wheat-Pst has been extensively conducted in the fields of histology, cytology, molecular biology, and biochemistry. However, due to special features of hexaploid wheat with large and complex genome and difficulties for transformation, and of Pst without clearly defined sexual reproduction and hard to culture on media, studies of genes involved in the wheat-Pst interactions using genetic and molecular techniques were largely limited. Therefore, deciphering the expression profiling of wheat-Pst interactions at the transcriptome level, along with identification and characterization of the resistance related genes, is of greatly importance to elucidate the molecular mechanisms of wheat-Pst interactions, which will be useful for development of genetically improved disease resistance and appropriate deployment of resistant cultivars to achieve sustainable control of the disease.In the study for my Ph.D. degree, expression profiling of wheat cultivar'Suwon11'infected by Pst pathotypes CY31 and CY23, representing compatible and incompatible reactions, respectively, were analyzed using the cDNA-AFLP technique, followed by isolation of the differentially expressed transcript derived fragments (DE-TDFs). Quantitative RT-PCR (qRT-PCR) analyses were used to verify the cDNA-AFLP patterns of selected important resistance-related genes. A wheat full-length cDNA library was screened using a PCR method, together with the bioinformatics characterization to identify resistance-related genes encoding kinases, pathogenesis related proteins and hypersensitive reaction induced genes. The biological functions of the identified candidate genes were determined using the BSMV-VIGS (Barley Stripe Mosaic Virus-Virus Induced Gene Silencing) approach. The follow most important results were obtained: 1. Suwon11 had compatible and incompatible reactions when infected with CY31 and CY23, respectively. Leaf samples were collected at each time point of 12, 18, 24, 36, 48, 72, 96, 120, 144, and 168 h after inoculation with Pst to extract total RNA and synthesize cDNA using reverse transcription. cDNA from each of the samples were used to determine differential expression profiling through cDNA-AFLP analyses. A total of 54,912 and 52,992 DE-TDFs from the compatible and incompatible interactions were detected, respectively, using 64 AFLP primer combinations, of which 33 produced reliable polymorphic bands. Expression profiling of 2,306 DE-TDFs in the compatible interaction showed differentially expressed (accounting for 4.2% of the whole compatible profiling), of which 1,340 DE-TDFs were up-regulated and 966 down-regulated. In the incompatible interaction, 2,437 DE-TDFs were differentially expressed (accounting for 4.6% of the whole incompatible profiling), of which 1,787 DE-TDFs were up-regulated and 650 down-regulated. From the characterized DE-TDF, 186 from the compatible interaction and 255 from the incompatible interactions were successfully cloned, sequenced, and assembled. A large portion of the DE-TDFs with unclear classification or no hits accounted for more than 60% in the compatible and 56% in the incompatible interaction. The majority of the DE-TDFs with function identified in each interaction were mainly involved in basal metabolism, signal transduction, and disease/defense groups. The results suggest that the host strengthens its metabolism and accumulates more nutrients to fulfill the wheat-Pst interactions. Additionally, based on their functional classification from both of the interactions, 47 genes were selected for further qRT-PCR analyses, along with their expression patterns in the mock-inoculated samples. The expression patterns revealed by qRT-PCR analyses were basically consistent with the cDNA-AFLP patterns. Because the expression changes of the genes in mock-inoculated samples were not significant, the identified DE-TDFs were most likely pathogen-induced genes.2. To compare genes in the compatible interactions with those in the incompatible interaction, BLASTN analyses were conducted to search against the database of DB-TDFs from the compatible interaction using the unigenes from the incompatible interaction as the query. The results showed that the 161 genes were shared by the two interactions. These genes were predicted to encode proteins involved in energy and metabolism, signal transduction and disease/defense, and protein metabolism. In contrast, 94 genes were unique in the incompatible interaction, which were also in the above mentioned categories. The results of qRT-PCR analysis to compare the expression patterns of selected shared and unique genes showed that most of the genes were induced in both of the incompatible and compatible interaction, but with different expression levels and at different time points between the two interactions. Therefore, we proposed that differentially expressed genes with different functional categories were most similar in two interactions, and the expression level or time of these genes might determine the resistance phenotype.3. Five kinase-encoding genes were obtained using the PCR-based method of screening the full-length cDNA library. These genes were TaRLK (2,393 bp), TaCIPK (1,993 bp), TaLRR-RLK (2,272 bp), TaSTK (1,831 bp), and TaCDPK (1,441 bp), encoding receptor protein kinase, CBL interacting protein kinase, LRR-receptor protein kinase, Ser/Thr protein kinase, and Ca2+ dependent protein kinase, respectively. The results of the InterProScan analyses characterized the five genes to encode kinases. TaRLK, TaCIPK, and TaSIK proteins were found to have an ATP-dependent phosphorylation feature. Kinases-like TaRLK and TaLRR-RLK contained N-terminus signal peptides, which might be helpful for secretion. TaCIPK and TaLRR-RLK each contained a trans-membrane domain. TaCIPK had a typical NAF motif of its protein family, encoding 25 amino acids in responsible of regulating the TaCIPK and CBL interaction. The analysis of multiple alignment and phylogenetic tree demonstrated that TaRLK, TaCDPK, TaCIPK, TaLRR-RLK, TaSTK were highly similar to kinase genes in wheat (AAK20741), rice (BAD53576), barley (ABQ09285 and ABF18544), and maize (NP₀01147794). Expression patterns revealed by the qRT-PCR analysis indicated that these genes, except for TaSTK, were induced with high levels of expression at the earlier time points during the incompatible interaction. The rapid accumulation of these genes in the early wheat-PST interaction might be a sign of implication to wheat defense response to Pst for these genes.4. Sixteen kinase-encoding DE-TDFs were selected for further functional characterization using the BSMV-VIGS technique. Positive silencing for each of the gene was detected using qRT-PCR. Of the 16 genes, 11 showed phenotypic changes on the plants with the genes silenced. Further histological observations supported the results. Plants with silenced genes including Su11_CY31₄885, Su11_CY31₅766, Su11_CY23_C304, Su11_CY23₂159, and Su11_CY23_C90 showed larger area of hypersensitive cell death with slight amount of sporulation (infection type 1+). Wheat plants with silenced Su11_CY31₁189, Su11_CY31_C193, Su11_CY31₃468, or Su11_CY23₂780 gene showed little sporulation around the small area of hypersensitive cell death, indicating a resistant reaction (infection type 2). Plants with silenced Su11_CY31₂485 or Su11_CY31₁437 gene showed a compatible reaction (infection type 3+) when being challenged with CYR23. The results indicated that Su11_CY31₄885, Su11_CY31₅766, Su11_CY23_C304, Su11_CY23₂159, Su11_CY23_C90, Su11_CY31₁189, Su11_CY31_C193, Su11_CY31₃468, and Su11_CY23₂780 are involved in wheat defense response against CYR23 with low-level effects, while Su11_CY31₂485 and Su11_CY31₁437 play a more important role in the defense response. In addition, compared with control plants without virus inoculation, plants inoculated by wild-type BSMV virus showed larger area of hypersensitive cell death after CYR23 inoculation, without sporulation, therefore indicating that virus may induce defense response as well.5. Three wheat pathogenesis-related (Pr) protein genes, TaPR1b (862 bp), TaPR5 (810 bp), and TaPR10b (975 bp), were successfully identified by screening the cDNA library and using the RACE technique. The encoded proteins of TaPR1b, TaPR5 and TaPR10b consisted of 164, 172, 166 amino acids, respectively. The TaPR1b and TaPR5 proteins were found to have a signal peptide within its N-terminal residues lacking of transmembrane, suggesting the putatively secretary function, while TaPR10b did not have a transmembrane or signal peptide structure. TaPR1b, TaPR5 and TaPR10b transcripts were induced upon Pst inoculation, with slightly higher transcription levels in the incompatible reaction than the compatible reaction, indicating that TaPR1b, TaPR5, and TaPR10b may be involved in wheat against the stripe rust fungus. Additionally, they were sensitive to SA and JA, and therefore, may play a role in resistance to Pst in the signal pathway of SA and JA. Transient expression analyses using gun-mediated bombardment method revealed that TaPR5 proteins were located in the epidermal cell walls. Immunocytochemical localization of TaPR5 in wheat-Pst interactions showed more gold particles were deposited over host cell wall in incompatible reaction, indicating that TaPR5 may take part in resistance to Pst.6. Five wheat genes, TaBI-1 (1,095 bp), TaDAD (608 bp), TaPrx (689 bp), TaELMO (1,146 bp), and TaLLS (1783 bp), were successfully cloned by screening the cDNA library and using the RACE technique. These genes were identified using a series of bioinformatical methods. To further characterize their functions, TaDAD2, TaBI-1, and TaLLS were silenced with the BSMV-based VIGS system. The results showed that conspicuous hypersensitive response (HR) was elicited by CYR23 on leaves pre-infected with BSMV:γor BSMV:TaDAD2as and BSMV:TaBI-1as, as well as mock-inoculated plants. However, limited fungal sporulation around the necrotic spots was observed only on leaves infected with BSMV:TaDAD2as and BSMV:TaBI-1as by 10 day post inoculation. Therefore, knocking down TaDAD2 or TaBI-1 expression reduced fungal-induced cell death and allowed limited fungal growth and uredium development, which indicate that TaDAD2 may function as a suppressor of cell death in the early stages of wheat-stripe rust fungus interaction. Additionally, necrotic cell areas were significantly smaller compared with BSMV:γand mock-inoculated treatments when TaLLS was knocked down. These results suggested that TaLLS was a positive regulator of plant cell death in wheat-Pst interaction.In conclusion, the results of this study have elucidated molecular mechanisms and advanced our understanding of wheat-Pst interactions. The genes identified to be related to resistance could be used to improve stripe rust resistance in wheat cultivars through transformation or genetic manipulation.