Progress 01/12/17 to 06/30/18
Outputs
Target Audience:This project provided training opportunities for one postdoc (on bioinformatic analysis), two graudate students (on gene cloning, protein localization, imaging), and one undergraduate student (on powdery mildew infection test). Also the genomic information obtained about strawberry powdery mildew will eventually help strawberry growers better control this fungal pathogen. Changes/Problems:We changed the objectives of Aim 2 and modified those of Aim3, based on the negative results from Aim1. This is because Aim2 (Determine if RPW8-like geneos indeed confer resistance to powdery mildew in F. vesca) is completely contiginent on Aim1 (Test if RPW8-like genes from strawberry can activate powdery mildew resistance iin Arabidopsis). Thus for Aim2, we decided to put our efforts in studying the fungal pathogens that causes powdery midlew disease on dicot plants including strawberry. We sequenced the genome and transcriptome of the strawberry powdery mildew. As for Aim3, in order to identify host nutrient transporters that might be manipulated by powdery mildew, we focused on identification of the causal mutations in Arabidopsis mutants that show resistance to powdery mildew in the absence of three essential immunity genes. We were able to identify two genes, one (MKP1) as a negative regulator a defense signaling pathway independent of salicylic acid and the other (MLO2) as a host susceptibility factor. Homologs of both genes in strawberry can be used for engineering powdery mildew resistance in strawberry in the future. What opportunities for training and professional development has the project provided?This project has provided training opportunities for one postdoc (Ying Wu), three graduate students (Harly King, Qiong Zhang and Bruce Levine) and one undergraduate student (Robin Denny) in bioinformatic, gene cloning, gene expression analysis and disease phenotypic characterization. How have the results been disseminated to communities of interest?Part of the results was included in a comprehensive manuscript on powdery mildew genome evolution recently published in BMC Genomics (Wu, Y., Ma, X., Pan, Z., Kale, S.D., Song, Y., King, H., Zhang, Q., Presley, C., Deng, X., Wei, C.I., and Xiao, S. (2018). Comparative genome analyses reveal sequence features reflecting distinct modes of host-adaptation between dicot and monocot powdery mildew. BMC Genomics 19, 705. https://www.ncbi.nlm.nih.gov/pubmed/30253736) What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
There are three objectives for this seed grant proposal. We completed Aim 1, modified the work for Aim 2 based on the results of Aim1, and partially completed Aim 3 and initiated a new Aim (Aim 4). Below is the detailed description of the experiments done and the results obtained. For Aim 1, we have cloned five strawberry RPW8-like genes (FvR8Ls) and made translational fusion with YFP at the C-termini of these genes in a binary vector that contain the RPW8.2 promoter to drive the expression of the R8L-YFP fusion genes. After sequence conformation, we transiently expressed these fusion genes in Nicotiana benthamiana and found that they were all nicely expressed. We then generated stable Arabidopsis (Col-0 accession) transgenic lines expressing each of these fusion genes and tested the T1 and T2 generation with a well-adapted powdery mildew isolate, Golovinomyces cichoracearum (Gc) UCSC1. However, none of the transgenic lines showed significant resistance despite powdery mildew induced expression. Also, none of the FvR8L-YFP proteins are localized to the extrahaustorial membrane (EHM) as the Arabidopsis RPW8.2-YFP. This result, though not entirely unexpected, is discouraging for pursuing further downstream work planned for Aim 2 and might suggest that either the FvR8L genes have minor contribution to powdery mildew resistance or they possess different functions in strawberry. For Aim 2 which is contingent to Aim 1, we decided to change the objective because of the negative result from Aim 1. We wish to investigate pathogenesis mechanisms of strawberry powdery mildew by sequencing the genome of Podosphaera aphanis (Pa) UMSG4, the powdery mildew isolate that infects strawberry. This fungus was identified in the greenhouse of USDA ARS Beltsville and has been maintained on diploid strawberry Fragaria iinumae that is very susceptible to Pa UMSG4. High quality genomic DNA and RNA was prepared and good quality of sequences have been obtained and sequenced, and the sequence data are being analyzed. In the same time, we also sequenced the genome of three other powdery mildew species that infect tommato, tobacco and sow thistle respectively for studying genome evolution of powdery mildew by comparative genome analysis. A research article on these data has recently been published. For Aim 3, we have been trying to definitively identify and characterize the causal mutations in 15 Arabidopsis mutants that display altered infection phenotype upon infection of both well-adapted or poorly adapted powdery mildew fungi including strawberry powdery mildew. So far, six causal mutations that result in enhanced resistance have been identified and confirmed by CRISPR-based targeted mutagenesis. One is in the MAP kinase phosphatase 1 (MKP1) gene and the remaining five are in the Mildew Locus O2 (MLO2) gene. MKP1 appears to be a negative regulator of plant resistance against powdery mildew that is independent of the EDS1/PAD4- and salicylic acid-dependent signaling, while MLO2 appears to be a host susceptibility factor of powdery mildew. Thus,the homologs of these two genes in strawberry could serve as promising target genes for engineering powdery mildew resistance in strawberry in the future. We have also initiated a new aim (Aim 4: genome assembly and gene annotation of strawberry powdery mildew) to obtain high-molecular weight DNA from the strawberry powdery mildew for PacBio sdequencing to improve geome sequencing and assembly for strawberry mildew. Once completed, strawberry and strawberry powdery mildew interestion can serve as a new model crop-mildew interaction system for studying host defense and fungal pathogenesis.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Wu, Y., Ma, X., Pan, Z., Kale, S.D., Song, Y., King, H., Zhang, Q., Presley, C., Deng, X., Wei, C.I., and Xiao, S. (2018). Comparative genome analyses reveal sequence features reflecting distinct modes of host-adaptation between dicot and monocot powdery mildew. BMC Genomics 19, 705. https://www.ncbi.nlm.nih.gov/pubmed/30253736
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Progress 01/12/17 to 09/30/17
Outputs
Target Audience:This project provided training opportunities for two graduate students and one undergraduate student to practise gene cloning from strawberry and plant transformation using the Arabidopsis model system. Changes/Problems:We made a change for Aim 2, because pursuing this aim should be based on positive results from Aim 1. Unfortunately, our results from Aim 1 showed that none of the five strawberry RPW8-like genes, when ectopically expressed in Arabidopsis, could confer significant resistance to powdery mildew. We thus devoted our efforts towards understanding the strawberry powdery mildew pathogen by obtaining and analyzing it genome and transcriptome. What opportunities for training and professional development has the project provided?This project has provided training opportunities for two graduate students (Harlan King and Bruce Levine) and one undergraduate student (Robin Denny) in gene cloning, gene expression analysis and disease phenotypic characterization. How have the results been disseminated to communities of interest?Part of the results from this project has been included in a comprehensive manuscript on powdery mildew genome evolution (under review by BMC Genomics). What do you plan to do during the next reporting period to accomplish the goals?1). We plan to analyze the DNA and RNA sequences from Pa UMSG4 and compare the gene content of this powdery mildew species to other dicot powdery mildew fungi and define its secretome and effectome. 2) We will continue to identify additional causal mutations that result in enhanced resistance or susceptibility in the Arabidopsis mutants. If more promsing genes (such as those that encode nutrient transporters or regulators) identified, we will perform targeted mutagenesis in strawberry to engineer resistance to powdery mildew.
Impacts
What was accomplished under these goals?
For Aim 1, we have cloned five strawberry RPW8-like genes (FvR8Ls) and made translational fusion with YFP at the C-termini of these genes in a binary vector that contain the RPW8.2 promoter to drive the expression of the R8L-YFP fusion genes. After sequence conformation, we transiently expressed these fusion genes in Nicotiana benthamiana and found that they were all nicely expressed. We then generated stable Arabidopsis (Col-0 accession) transgenic lines expressing each of these fusion genes and tested the T1 and T2 generation with a well-adapted powdery mildew isolate, Golovinomyces cichoracearum (Gc) UCSC1. However, none of the transgenic lines showed significant resistance despite powdery mildew induced expression. Also, none of the FvR8L-YFP proteins are localized to the extrahaustorial membrane (EHM) as the Arabidopsis RPW8.2-YFP. This result, though not entirely unexpected, is discouraging for pursuing further downstream work planned for Aim 2 and might suggest that either the FvR8L genes have minor contribution to powdery mildew resistance or they possess different functions in strawberry. For Aim 2 which is contingent to Aim 1, we decided to change the objective because of the negative result from Aim 1. We wish to investigate pathogenesis mechanism of strawberry powdery mildew by sequencing the genome of Podosphaera aphanis (Pa) UMSG4, the powdery mildew isolate that infects strawberry. This fungus was identified in the greenhouse of USDA ARS Beltsville and has been maintained on diploid strawberry Fragaria illumae that is very susceptible to Pa UMSG4. High quality genomic DNA and RNA has been prepared and good quality of sequences have been obtained and are under analysis. For Aim 3, we have been trying to definitively identify and characterize the causal mutations in 15 Arabidopsis mutants that display altered infection phenotype upon infection of both well-adapted or poorly adapted powdery mildew fungi. So far, one causal mutation that results in enhanced resistance has been confirmed by CRISPR-based targeted mutagenesis to be in MAP kinase phosphatase 1 (MKP1). MKP1 appears to be a negative regulator of plant resistance against powdery mildew that is independent of the EDS1/PAD4- and salicylic acid-dependent signaling. Thus, functional MKP1 homologs may be a promising target for future engineering powdery mildew resistance in strawberry.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
Comparative genome analyses reveal sequence features reflecting distinct modes of host-adaptation between dicot and monocot powdery mildew
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