Virus composition
Next-generation sequencing was employed to characterize viruses and virus-like particles present in the canes of two wine grape cultivars, Merlot and Syrah. Three symptomatic vines from each cultivar from commercial vineyards that showed characteristic reddish-purple foliar coloration at the post- véraison stages, indicative of viral infections, were employed in the study while asymptomatic vines were chosen that are adjacent to the symptomatic vines to minimize spatial variation. The sequencing reads post adapter trimming and quality control were mapped to GenBank (viral 1.1 genomic) in CLC Genomics Workbench 8 platform. With an average of 45.75 M paired reads, of which 3.83% were mapped to viruses and virus-like sequence database (Table 1). All the symptomatic vines from both Merlot and Syrah cultivars contained Grapevine red blotch virus (GRBV), while none of the sequence reads corresponding to asymptomatic vines were mapped to Grapevine red blotch viral genomic sequences (Table 1). The additional viruses found in all samples of both cultivars were Grapevine Fanleaf Virus and Rupestris Stem Pitting Virus. The deep sequencing data was confirmed by the RT-PCR analysis (Figure S1). Three symptomatic and two asymptomatic samples from Syrah cultivar contained Grapevine Syrah Virus, while only two asymptomatic Merlot vines had this virus. Similarly, two viroids, Hop StuntViroid and Grapevine Yellow Speckle Viroid were present in all samples, with greater sequencing depth and complete genome coverage.
Carbohydrate analysis
At the commercial wine grape harvest, the berries from symptomatic and asymptomatic vines of Merlot and Syrah were analyzed for berry sugar content and the values are expressed as 0Brix. The results indicate that the 0Brix of berries from symptomatic vines were significantly reduced in both cultivars (Figure 1a). Soluble sugars (glucose, fructose, and sucrose) and starch analysis in mature leaves showed that sucrose and starch concentrations were significantly elevated in symptomatic vines of cv. Merlot whereas starch was accumulated significantly in cv. Syrah, relative to the asymptomatic counterparts (Figure 1b, c). This observation implies an alteration of the source-sink relationship due to virus infection. To understand how these changes reflects structural carbohydrates, we analyzed the structural cell wall carbohydrates and lignin contents. The HPLC analysis of acid-soluble cell wall fraction identified arabinose, galactose, glucose, mannose, rhamnose, and xylose (Table 2), and a comparison of these sugars between symptomatic and asymptomatic vines did not show significant differences. A slight increase in arabinose was apparent in the symptomatic vines of Syrah relative to their asymptomatic counterparts (Table 2). The total Klason lignin contents (acid-soluble and acid-insoluble) were estimated to be approximately 33 and 30 percent in Merlot and Syrah canes, respectively. No significant differences were found in lignin levels between symptomatic and asymptomatic vines of both cultivars. Collectively, the results from structural carbohydrate analysis suggest that GRBV infection did not alter the cell wall composition of GRBV infected grapevine canes, whereas the soluble sugars and starch contents were elevated in source leaves with concurrent reduction of berry sugars.
Glycome analysis
Glycome profiling analyses were conducted on cell walls fractions isolated from the cane woods of asymptomatic and symptomatic Merlot and Syrah cultivars (Figure 2). Overall, the cell wall glycome profiles were largely similar irrespective of the cultivars or status of viral infection. Glycome profiling studies revealed the overall composition and extractability of diverse matrix cell wall glycans present in all samples studied. In general, xyloglucan epitopes were extracted in all alkaline extracts such as carbonate, 1M KOH, 4M KOH, and 4M KOHPC extracts. Further xylan epitopes (both substituted and unsubstituted xylans) were extracted out in all samples except the oxalate extracts. Pectic backbone epitopes (recognized mainly by HG-backbone-1 and RG-I backbone groups of mAbs) were significantly present in all cell wall samples and followed a mostly identical pattern of extractability. Pectic arabinogalactan epitopes (recognized by RG-I/AG, and AG-1 through 4 groups of mAbs) were also significantly present in all samples, however; their patterns of extractability varied among two varieties. For instance, a marginal increase in the extractability of pectic arabinogalactan epitopes was observed in oxalate and carbonate extracts of Syrah samples compared to Merlot. Subtle variations in the extractability of pectic arabinogalactan epitopes were also noted across asymptomatic and symptomatic samples within a given cultivar. For instance, a reduced abundance of pectic arabinogalactan epitopes was observed in the oxalate and carbonate extracts from symptomatic samples from both cultivars hinting that the viral infection caused cell wall alterations. To further study underlying variations we conducted more in-depth statistical analyses using the raw data as described in the next section.
The glycan extractability of asymptomatic Merlot and Syrah clearly showed varietal differences in cell wall matrix complexity. The antibody binding data was filtered (low-pass binding values were removed) and the remaining immune data was clustered into data self-organizing maps (SOM-clustering). Pectic polysaccharide glycans (epitopes of homogalacturonan backbone and rhamnogalacturonan-1/ arabinogalactans) were extracted at higher levels in the initial mild extracts (Figure 3, cluster 1 and 4) of Syrah compared to Merlot. The second variation was observed in non-fucosylated xyloglucan epitope extractability, with relatively higher levels being recovered during oxalate, carbonate, and 1M alkali extracts of Merlot than in Syrah. The remaining antibody signal intensities did not vary between two cultivars suggesting any further changes in their cell wall composition were unlikely.
When we compared the differences in the cell wall extractability of virus-free and virus-infected vines, two changes were consistent in both cultivars. First, loosely bound pectic arabinogalactan epitope abundances were reduced in the GRBV infected grapevines. These changes were especially observed when the cell wall fractions were extracted with sodium carbonate (SC). The reduction in the extractability was more pronounced in the Syrah cultivar (Figure 3, cluster 4). In the subsequent extracts, we did not observe any detectable changes between GRBV-free and GRBV infected cane samples. Second, GRBV-infection resulted in a specific increase in extractability of non-fucosylated xyloglucan epitopes in stronger alkaline extracts (4M KOH and 4M KOHPC), and this was relatively more prominent in Syrah compared to Merlot. This increase was observed in the 4M KOH extracts in both cultivars; however, during the strongest alkali extraction (with 4M KOHPC) the Syrah samples showed higher extractability compared to Merlot. In addition to these changes, GRBV infected Syrah exhibited lower xylan epitope extractability in relatively stronger alkali extracts (1M and 4M KOH). The remaining samples did not show any changes in their extractability. The least affected group of glycan epitopes contained those of galactomannans, epitopes recognized by rhamnogalacturonan-1b group of mAbs, and epitopes recognized by the reminder of the arabinogalactan groups of mAbs (Figure 3, cluster 3). The glycome analysis thus provided insights into how different cell wall carbohydrates are compositionally and structurally packaged in cane woods of wine grapes, and how virus infection changes some of the cell wall architecture compared to their virus-free counterparts.