PA treatment induces genome-wide transcriptional changes consistent with IR
To gain insights into the molecular mechanisms behind PA-IR, mRNA from roots and shoots of PA-treated tomato plants was sequenced. By sequencing root and shoot samples, both local responses (shoots) and systemic (roots) transcriptional responses were analyzed. Principal component analysis and hierarchical clustering showed that PA treatment led to substantial transcriptome shifts in both tissues (Figure 3 ). When comparing PA-treated and mock-treated plants, 164 genes were differentially expressed (DE) in shoots (133 up, 31 down) and 309 in roots (86 up, 223 down). A full list of transcripts and their expression changes can be found in Supplementary Table S2.
Local transcriptional response to PA treatment
Gene ontology (GO) analysis identified 69 enriched GO terms in PA-treated shoots compared to mock-treated shoots, of which 23 were related to immunity (e.g., systemic acquired resistance andregulation of immune response; Supplementary Table S3 ), 11 to abiotic stress and four to photosynthesis. InterPro domain analysis also identified enrichment of immunity- and photosynthesis-related domains (Supplementary Table S3 ).
Among immunity-related genes, 15 genes annotated as encoding pathogenesis-related (PR) proteins were DE in PA-treated shoots, all of which were upregulated. Some of these have been used as IR markers in tomato, e.g. PR1a (Schuhegger et al. 2006; Martínez-Medinaet al. 2017). Other genes involved in IR are also upregulated in shoots after PA treatment, including four NIM1-INTERACTING2-like (NIMIN2-like) genes and the tomato ortholog of Arabidopsis thaliana SAD4 (SAR-DEFICIENT 4). NIMIN2 is an interactor of NPR1 (Nonexpressor of PR genes) and has a role in IR establishment (Zwicker, Mast, Stos, Pfitzner & Pfitzner 2007), and SAD4 is involved in the biosynthesis of the immunity regulator L-pipecolic acid (Shan & He 2018). The effect of PA on shoot immunity is further reflected by the induction of six genes encoding WRKY transcription factors.
Both NIMIN2-like and PR1a are SA-inducible, but no genes known to be involved in SA biosynthesis were DE. There were no clear expression changes in genes involved in abscisic acid, auxin or jasmonic acid biosynthesis and signaling, but two1-aminocyclopropane-1-carboxylate oxidase-like genes possibly involved in ethylene biosynthesis were upregulated in shoots.
PA also appears to affect shoot reactive oxygen species (ROS) metabolism, as four genes encoding PEROXIDASES and six encoding GLUTHATIONE-S-TRANSFERASES were upregulated.
Although PA is a well-characterized C4H inhibitor (Schalk et al.1998; Schoch et al. 2002), our data show no change in shoot expression of C4H , nor in that of PAL or 4CL, two other early phenylpropanoid pathway (PPP) gene families. However, several downstream PPP-related genes were upregulated: seven genes involved in flavonoid biosynthesis and glycosylation (two putativeFLAVANONE-3-HYDROXYLASES, one putativeISOFLAVONE-2’-HYDROXLYASE and fourUDP-GLYCOSYLTRANSFERASES acting on flavonoids, includingTOMATO WOUND-INDUCED 1 ) and three genes involved in lignification and cell wall reinforcement (Caffeoyl-CoA-O-methyltransferase 1 , a cinnamyl alcohol dehydrogenase and THT1-3, an N-hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyl transferase) . Several other genes involved in cell wall development were also upregulated, including a CELLULOSE SYNTHASE and twoGLYCINE-RICH CELL WALL PROTEINS. Together, these results suggest active cell wall remodeling upon PA treatment.
Systemic transcriptional response to PA treatment -
To investigate the effect of foliar PA application on gene expression in systemic (untreated) tissues, mRNA-sequencing was performed on roots of PA-treated tomato plants. GO analysis of DE genes in roots is consistent with an IR phenotype, 20 out of 45 enriched GO terms were related to plant immunity and five to abiotic stress response (Supplementary Table S3 ).
As in shoots, foliar PA treatment strongly induced PR gene expression in roots: 16 genes annotated as possible PR genes were DE, of which 14 were upregulated. Again as in shoots, these PR genes include IR markers such as PR1a . ROS metabolism might also be altered in roots upon PA treatment: four PEROXIDASES , one LACCASE and oneCATALASE (but no GLUTHATIONE-S-TRANSFERASES) were upregulated.
In contrast to PR- and ROS-related genes, WRKY and ERF transcriptions factors show a divergent expression pattern between root and shoot after PA treatment. While five WRKY genes were upregulated in shoots after PA treatment, seven were downregulated in roots and none upregulated. Genes encoding ETHYLENE RESPONSE FACTORS (ERFs), another major class of transcription factors involved in biotic stress response, also show a contrasting expression pattern between root and shoot: in shoots one such gene is upregulated, whereas in roots nine ERFgenes were downregulated and three upregulated.
The expression of PPP and cell wall remodeling genes upon PA treatment also differs between shoot and root. One PAL gene (Solyc09g007900.5) is downregulated in roots after PA treatment, but no other early PPP genes are DE. One Hydroxycinnamoyl-CoA-quinate transferase involved in chlorogenic acid biosynthesis and oneSINAPATE GLUCOSYLTRANSFERASE were upregulated, but – in marked contrast to shoots – no genes related to flavonoid biosynthesis were DE. Several cell wall-related genes were DE in roots of PA-treated plants: two cellulose synthases and THT1-3 were downregulated, whereas Xyloglucan endotransglucosylase/hydrolaseand two pectinesterases were upregulated.