The four others were an autophagy gene and constitutively activated cell death 1 , which function in autophagy, lytic pore formation, and HRs , Kinesin-like 5C , which encodes a microtubule motor protein , and an ARF-GAP encoding ADP-ribosylation factor GTPase-activating protein domain 15, which helps efficiently load vesicles and remodel the actin cytoskeleton . Several additional general functional categories were present among the 32 genes that exhibited conserved responses to GLRaVs . Genes encoding phenylalanine ammonia-lyase and cinnamate 4-hydroxylase , which catalyse the first two steps of the phenylpropanoid pathway, two genes encoding UDP glucosyltransferases , which conjugate sugars, and SWEET17, encoding a sugar transporter, were upregulated, as was a gene encoding 3-isopropylmalate dehydratase, an enzyme in the leucine biosynthetic pathway. Two genes, encoding an LRR receptor-like kinase called ERECTA and nicotianamine synthase , were downregulated. ERECTA participates in organ development and resistance to bacterial and fungal pathogens . NAS expression increases Fe and Zn abundance in rice . Generally, these genes and their changes in expression suggest that a common response to GLRaVs in Cabernet Franc berries during ripening includes the modulation of pathogen-detecting genes, an increase in ABA transport and signalling, a decrease in ROS-related signalling, and an enhancement of cytoskeleton remodelling, vesicle trafficking, phenylpropanoid metabolism, sugar transport and conjugation, and leucine biosynthesis.The same berry samples used for RNA-Seq were used to measure the levels of three hormones associated with ripening and/or stress, including SA, JA, and ABA, and additional metabolites, square plastic plant pot including xanthoxin, a precursor to ABA, and ABA glucose ester , a conjugate of ABA implicated in its long-distance transport .
The mean levels of SA and JA were significantly influenced by year and/or by interactions between year, rootstock, and GLRaV at prevéraison , but no significant differences were observed between individual groups . In contrast, year alone had a significant impact on the levels of ABA and related metabolites measured at each developmental stage, but largely did not interact with rootstock or GLRaV infection type to affect the abundance of ABA and related metabolites . In addition, the effect of GLRaV infection on ABA and ABA-GE content significantly differed based on rootstock . Significant differences in ABA and ABA-GE content were observed between rootstocks in plants with identical infection status and between plants with different GLRaV status grafted to the same rootstock . Such differences were scarcely observed for xanthoxin, a precursor to ABA . Significant differences between rootstock genotypes in the abundance of these metabolites were observed most at prevéraison and in GLRaV , GLRaV-3 , and dual infections . In GLRaV and most single infection conditions, the levels of all three metabolites tended to be higher in berries from plants grafted to MGT 101-14 than in berries from plants grafted to Kober 5BB. The opposite tended to be true when two GLRaVs were present. With one exception, significant changes in the abundance of ABA and ABA-GE in GLRaV versus GLRaV were typicallyto cluster separately from other Kober 5BB-grafted plants. Nonetheless, the effects of GLRaVs differed between rootstocks for many of these genes . One of these, encoding an ABC transporter , is also included in Figure 3; significant increases in its expression were observed in both years, in both rootstock conditions, and for several GLRaV infections. All other significant changes in GLRaV versus GLRaV that were reproduced in both years occurred in only one rootstock or the other.
These changes were sparse. However, significant differences between rootstocks in identical GLRaV were reproduced in both years for 9 out of these 19 genes. Significant differences between rootstocks in at least one year were observed for 16 out of these 19 genes. On average, three genes encoding 9-cis-epoxycarotenoid dioxygenases , both ABA 8′-hydroxylase genes, three ABC transporter genes, one gene encoding PP2C, and PYL/RCAR were upregulated in berries from plants infected with GLRaV in both rootstock conditions. One ABC transporter gene was downregulated in both rootstock conditions. Of the remaining eight genes, most were downregulated across development only in berries from MGT 101-14-grafted plants.Differential expression analysis identified 1,809 genes that were differentially expressed in at least 1 year, that were differentially expressed in only one rootstock condition and more than one GLRaV infection type versus GLRaV , and/or for which the effects of more than one GLRaV infection significantly differed between rootstocks . RNA-Seq, hormone, and metabolite data from ripening Cabernet Franc berries were integrated in a multiple factor analysis to relate these variables and distinguish the effects of GLRaVs given different rootstocks . As input for the MFA, all genes differentially expressed between GLRaV and GLRaV or between rootstocks were used, plus all hormones and metabolites measured. Overall, the rootstocks were distinct at each developmental stage . Some of the GLRaV conditions could be distinguished from others at prevéraison, véraison, and harvest. At prevéraison, GLRaV-1,2 differed overall from GLRaV-1 . At véraison, GLRaV-1,3 differed from every other GLRaV condition except GLRaV-1,2 . At harvest, the two dual infections were different than one another and GLRaV-1,2 differed from GLRaV-1 . Next, we identified which variables were best correlated with each rootstock-differentiating MFA dimension. At eachdevelopmental stage, ABA and/or ABA-related metabolites were correlated with at least one of the first two MFA dimensions .
The rootstock-dependent disparity in ABA levels and ABA-related gene expression is consistent with the observation that ABA and related metabolites tended to be highly correlated with rootstock-differentiating MFA dimensions over time and that ripening initiates earliest in Kober 5BB plants with dual infections in terms of TSS . There were 548 genes that shared high correlation to rootstock-differentiating MFA dimensions with hormones or hormone-related metabolites. Most of these genes had shared positive or negative correlations to the same dimensions as ABA, xanthoxin, and/or ABA-GE . Categories of genes with functionally relevant relationships to each hormone or hormone related metabolite were over-represented among the genes that shared high correlation to rootstock-differentiating MFA dimensions with each hormone. ABA signalling, starch biosynthesis and catabolism, and C2C2-DOF transcription factor-encoding genes were over-represented among the genes correlated to the same dimensions as ABA and xanthoxin. The latter two categories were significantly over-represented among the genes correlated with the same dimensions as ABA-GE. Genes related to heat shock protein -mediated protein folding, chaperone-mediated protein folding, the cation channel-forming HSP-70, channels and pores, the reductive carboxylate cycle, and carbon fixation were over-represented among those correlated with the same MFA dimensions as SA. Similarly, most ripening-related metabolites measured were correlated with the same MFA dimensions as ABA, xanthoxin, and/or ABA-GE . Overall, the effects of GLRaVs differ between rootstocks primarily in terms of ABA and related metabolites. This finding is especially salient because of the role that ABA plays as a ripening promoternear véraison, in root–scion communication, and in plant stress. ABA, metabolites, and genes that were well correlated to rootstock differentiating MFA dimensions and were differentially expressed were scrutinized more closely.There were 548 genes in 85 functional categories that were well correlated with rootstock-differentiating MFA dimensions and differentially expressed between GLRaV grafted to different rootstocks or in GLRaV versus GLRaV in only one rootstock condition . These functional categories were generally related to hormone and other types of signalling, amino acid and other metabolic pathways, transcription factors, transport, and cellular organization and bio-genesis . Most of these genes coincided with ABA and related metabolites along rootstock-differentiating MFA dimensions . The distribution of expression for four transcription factor families differed significantly between rootstocks at all four developmental stages . This included bHLH, C2C2-DOF, FHA, 25 liter sqaure pot and homeobox domain transcription factors. The distribution of expression of 39 hormone signalling related genes differed significantly between rootstocks . This was true at each developmental stage for ABA, gibberellin , and auxin signalling genes and at three developmental stages for JA/SA, cytokinin , and ethylene signalling genes . Genes related to all of these hormone families had similar roles in MFA and were associated with ABA, including the JA/SA signalling genes .
This may reflect interactions between hormone signalling pathways. In addition, the effects of GLRaV infections on histone H1 expression were not equal in plants grafted to both rootstocks . Linker histone H1 contributes to higherorder chromatin structure . There were seven ABA signalling pathway genes that differentiated GLRaV effects in plants grafted to different rootstocks . This included SOS2, KEG, three PP2C genes , and two genes encoding ABA-responsive element -binding proteins . SOS2 is a kinase appreciated for its role in the salt stress response, seed germination, GA signalling , and ABA signal transduction via its interaction with ABI2 and ABI5 . SOS2 was upregulated in both rootstock conditions before and at véraison and downregulated after véraison. KEG is a negative regulator of ABA signalling; it maintains low levels of ABI5 in the absence of stress by ubiquitination and degradation and helps regulate endocytic trafficking and the formation of signalling complexes on vesicles during stress . KEG was downregulated in Kober 5BB and downregulated in MGT 101-14 at véraison and mid-ripening. In the presence of ABA, ABA receptors bind PP2Cs like HAB1 and AHG3 to inhibit their phosphatase activity. As a result, ABA signal transduction is permitted via SnRK2 phosphorylation of ABRE-binding proteins . ABI5 and AREB2 are bZIP transcription factors that bind to ABREs to drive ABA signalling and ABI5 can integrate signals across hormone signalling pathways . The effects of GLRaVs on these genes in Kober 5BB were consistent with an enhancement of ABA signalling during ripening. In Kober 5BB, HAB1, AHG3, AREB2, and ABI5 were upregulated. In MGT 101-14, the PP2Cs were downregulated at two or more developmental stages; AREB2 and ABI5 were upregulated at and after véraison.In addition to analysing hormones and hormone-related metabolites, we analysed metabolites associated with the shikimate, phenylpropanoid, and flavonoid pathways and their biosynthetic and regulatory genes in Cabernet Franc berries during ripening . Significant differences in expression versus GLRaV were detected, as well as significant differences in the effects of GLRaV infection between different rootstock conditions . The effects of GLRaV infection on the genes associated with this pathway were generally consistent with the change in abundance of corresponding metabolites . Overall, these genes tended to be upregulated in GLRaV at véraison . After véraison, the amount of upregulation tended to decrease, or genes were downregulated . The three amino acids examined tended to be less abundant in GLRaV across the developmental stages and the largest decreases were observed at harvest . Mixed effects of GLRaVs were observed on the abundance of hydroxycinnamic acids , t-resveratrol, and anthocyanins. Significant changes versus GLRaV tended to occur in only 1 year. During ripening, these were significantly more abundant in Kober 5BB GLRaV-1,2 , Kober 5BB GLRaV-4 , and/or MGT 101-13 GLRaV-3 . Significant decreases were observed for GLRaV-1 and GLRaV-1,3 . Though nonsignificant, the size of the downward effect of some GLRaV infections on these metabolites tended to increase towards harvest. Finally, flavanols and flavonol glycosides tended to be elevated in GLRaV . The size of this effect tended to be greatest before and at véraison and decreased towards harvest. Significant differences between rootstocks were observed for GLRaV-1,2 in both years and for GLRaV-1 , GLRaV-1,3 , and GLRaV-3 in individual years;the increase in flavonols and flavanols tended to be greater in berries from Kober 5BB GLRaV than in those from MGT 101- 14 GLRaV .GLRaVs affect viticulture on nearly every continent and can a have considerable economic impact on a major crop. The presence and severity of symptoms in GLRaV infected grapevines is influenced by host genotype, rootstock, which GLRaV is present, and environmental conditions. In addition to the assembly and annotation of the Cabernet Franc genome, a valuable resource that might be applied for the larger purpose of understanding grapevine genomic diversity and evolution, the dedicated experimental vineyard used in this study is a tremendous asset for the study of GLRaV infections over time in a common environment. This work identified responses to GLRaVs in grape berries during ripening, including those that are conserved across experimental conditions and responses that differ based on the rootstock present. We propose which hormones and signalling pathways at least partially govern the responses observed and likely influence leafroll disease symptoms. The effects of dual infections, particularly GLRaV-1,2 , were most distinctive.