Integrating Systems Biology with Marker Assisted Selection to Guide the Stacking of Powdery Mildew Resistance Genes

The long-term goal of this project is to develop grape varieties that possess effective and durable resistance to powdery mildew (PM). Stacking resistance genes from multiple resistant genetic backgrounds and with the least functional redundancy is a proven breeding strategy to improve both durability and level of resistance. This strategy requires (a) the identification of multiple sources of resistance, (b) the functional characterization of the mechanisms of resistance to prioritize optimal genetic combination, and, finally, (c) marker assisted breeding to introduce the selected genes into elite varieties.

In continuation of our multi-year breeding effort, with the awarded budget in the 2016-2017 funding period we have continued the functional characterization of resistance responses activated in presence of known powdery mildew resistance loci. The experiments for the functional characterization of Ren2, Ren3, and Ren4 were completed and data analysis is ongoing. We started the experiments for the functional characterization of Run1, Run1.1, Run2.1, and Run2.2. The manuscript describing the genetic analysis of Ren6 and Ren7 and the associated markers used for introgression and stacking was published in BMC Plant Biology (Pap et al, 2016).

Deep Sequencing for Trunk Disease Diagnostics

The aim of this multi-year project is to develop rapid and cost-effective diagnostic methods based on Next Generation Sequencing (NGS) technologies for detection, identification and quantification of trunk pathogens in asymptomatic and symptomatic grape wood. In the 1st year of the project (2015 – 2016) we collected diseased wood material from commercial vineyards and characterized the associated fungal pathogen species using traditional methods, such as morphological and sequence-based identification of purified fungal colonies (see progress report for the 2015-2016 funding cycle). We used these samples to determine how effective ITS-sequencing, meta-genome sequencing and meta­transcriptome sequencing approaches are in identifying and quantifying pathogenic species directly in planta. Data simulations allowed us to determine what mapping algorithm was the most specific and sensitive in detecting trunk pathogens both qualitatively and quantitatively. All NGS methods we tested were in agreement with traditional diagnostic methods, but also allowed us to detect simultaneously multiple pathogen species with no need of hands-on sample culturing and colony purification. Additionally, unlike traditional diagnostics, which are strictly qualitative, NGS approaches allowed us to determine the relative abundances of the different infecting species. Among all methods tested, ITS-seq is still the most cost-effective until library preparation costs for RNA and DNA-seq do not decline significantly. For this reason, ITS-seq was chosen for further protocol optimization. Both sensitivity and specificity of the ITS-seq approach remain to be improved for diagnostics purposes. In the second year of the project (reported here), we (a) confirmed that NGS allows the detection with high specificity of actively infecting pathogens when vines are experimentally infected with individual pathogen strains; (b) established that NGS detection is quantitative and allows to differentiate between diseased and healthy vines; (c) developed a protocol for testing dormant cuttings and started testing cuttings provided by a commercial nursery. In the 2016-2017 funding cycle, we also developed a new DNA extraction protocol that reduced the time required for processing and the amounts of sample, reagents and waste.

Deep Sequencing for Trunk Disease Diagnostics

The aim of this multi-year project is to develop rapid and cost-effective diagnostic methods for detection, identification and quantification of trunk pathogens in asymptomatic and symptomatic grape wood. In the 2015 -2016 grant cycle, we have tested different NGS-based methods for the detection of trunk pathogen in symptomatic infected field material and compared the results with traditional diagnostic approaches. All methods we tested were in agreement with traditional diagnostic methods. NGS-based methods also detected simultaneously multiple pathogen species with no need of tedious and hands-on sample culturing and colony purification. Additionally, unlike traditional qualitative diagnostics, deep sequencing also allowed to determine the relative abundance of the different species. Among all methods tested, ITS deep sequencing (ITS-seq) remains the most cost effective until library prep costs for DNA and RNA sequencing do not decline significantly. For this reason, ITS-seq will be the subject of protocol optimization of the second year of the project. Both sensitivity and specificity of the method need to be improved for diagnostics purposes. The results of this first phase of the project demonstrate that unlike traditional approaches NGS-based detection delivers rapid simultaneous identification and quantification of multiple species in infected tissue with no need of culturing and isolation.

Characterization of Genes Involved in the Grapevine’s Defense Response

We collected berry samples at several stages in berry development. The deformability, brix, berry number and the berry weight was recorded to document the developmental stage. Soluble proteins were extracted from the fruit at each stage and separated by gel electrophoresis. The corresponding western blots are being used to evaluate the relative abundance of two malic enzyme isoenzymes during ripening. We made three plasmid constructs using cDNA clones from two grape malic enzymes (VvMEl and VvME2) that were used for expression of the respective proteins in E. coli cultures. One construct was a full length cDNA for VvMEl, the second was an unprocessed full length cDNA for VvME2, and the third was a VvME2 that had been truncated 50 amino acids from the start methionine to give a version of VvME2 that is thought to be present in grape tissue after import of the polypeptide into plastids. The constructs were successfully transformed into an E. coli strain and results show that malic enzyme activity in crude extracts of the bacterial cells induced with IPTG increases over the uninduced cells. Thus, we have successfully introduced the grape malic enzyme cDNAs into E. coli and obtained expression of the active enzyme protein. In the current year we used a grape berry cDNA library to identify 10 genes that are expressed specifically at the beginning of ripening, and half of them correspond to proteins with known functions. One of the first genes identified is nearly identical to an enzyme that catalyzes rotation of peptide proline bonds (peptidyl-prolyl cis-trans isomerase). This enzyme aids polypeptide folding for proper conformation after synthesis of proteins in the cell. The second gene is very similar to a putative surface protein from Arabidopsis. Of the 158 amino acids in the open reading frame 113 were identical, and if conservative substitutions are considered 126 of the 158 matched. The third gene is nearly identical to ribosomal protein L10 (Large subunit, protein 10) from Arabidopsis. In an 86 amino acid segment, 72 were identical to the protein from Arabidopsis, and 20 other matches were found with ribosomal proteins from various organisms such as humans, pigs, yeast and rats. The fourth gene is very interesting and turned out to be a cap-binding protein. These are proteins that specifically recognize the 7-methyl guanosine group at the 5′ end of eukaryotic rnRNAs. The one we have identified from grape most closely matches a cap-binding protein from wheat called eIF4E (eukaryotic initiation factor 4E). The precise role of this protein in plants is not known, although work in Arabidopsis is currently underway in other laboratories and should soon reveal how eIF4E influences mRNA translation in plant cells. Another gene we identified is a malate/oxoglutarate transporter nearly identical to one reported from potato. There was also homology with a malate/oxoglutarate transporter from rice and Panicum miliaeum. These transporters are known to be located in the membranes of vacuoles, plastids and mitochondria where they regulate movement of malate and oxoglutarate in and out of these organelles. Thus, of the 10 genes we isolated, we were able to assign function to 5. The remaining 5 have no clear homology to proteins in the databases, but more sophisticated search strategies are expected to identify functional domains of these proteins which may suggest the function they have in vivo.