2.4 Root zone warming induces tomato immunity
Our results indicate that tomato pathogen-resistance is systemically modulated by RZW, as previously suggested (Elad et al., 2017, 2016a; Elad, 2018). To examine whether the decrease in fungal and bacterial disease following RZW is paired with increased plant defense in tomato, we tested known hallmarks of immune system activation: ethylene (C2H4) and ROS production and defense gene expression. Warmed WT M82 plants exhibited an increase in ethylene production (Figure 6a ), while the warmed plants of the Brigade cultivar did not exhibit a significant increase in wounding ethylene (Supplemental Figure S2 ), perhaps due to differences in the innate immunity mechanisms among these two cultivars as indeed, cv. Brigade plants are more susceptible to Bc than M82 plants.
To examine whether RZW augments defense responses elicited by known elicitors of plant defense, we employed the Xyn11 family xylanase Ethylene Inducing Xylanase (EIX), that induces Effector Triggered Immunity (ETI) in responsive cultivars (Sharon et al., 1993; Leibman-Markus et al., 2017; ; Bar and Avni, 2009), and the bacterial flagellin derived peptide flg-22, that is known to broadly induce immunity and ROS production (Felix et al., 1999; Segonzac and Zipfel, 2011). The combination of RZW and EIX or flg-22 induces immune responses- ethylene and ROS respectively, at greater levels than the elicitor alone in both cases (Figure 6a,b; Figure S2 ).
To analyze the alterations to tomato gene expression caused by RZW, we applied both 24 hour and 7 day RZW to M82 plants, and examined the expression of several known defense genes. Warming induced the expression of proteinase inhibitor 2 (PI-2 , Solyc03g020080), pathogenesis-related proteins (PR1a , Solyc01g106620) andPR-1b (Solyc00g174340), Pto-interacting 5 (Pti-5,Solyc02g077370), 1-aminocyclopropane-1-carboxylate oxidase 1 (ACO-1 , Solyc07g049530) and WRKY75 (Solyc05g015850) (Figure 7a ).
We also examined the effect of RZW on the expression of Pattern Recognition Receptors (PRRs) in the plant shoot. A recent report has demonstrated that increases in PRR expression correlate with enhanced immune outputs, being sufficient- along with cell damage- to mount strong localized immune responses to invading pathogens (Zhou et al., 2020). Figure 7b demonstrates that several PRRs are induced by RZW, indicating that PRR induction could underlie part of the induced resistance observed upon RZW.
We examined defense gene expression 24 hours after Bcinoculation, in both warmed and unwarmed plants (Figure 8 ). In most cases, Bc induced greater levels of the defense genes assayed when compared with RZW, consistent with the idea that the warming treatment causes immunity priming (Figure 8- compare black and pale-gray bars). For most assayed genes, RZW prior to B. cinereainoculation did not result in significant alterations to defense gene expression, when compared with Bc alone (Figure 8- black vs dark gray bars), suggesting that induced resistance by RZW decreases subsequent disease levels irrespective of its effect on gene expression. Positive correlations between B. cinerea disease levels and defense gene expression were reported previously (Meller-Harel et al., 2014; Mehari et al., 2015). The chosen genes are all hallmarks of pathogen responses (Martínez-Medina et al., 2013; Ament et al., 2004; Iberkleid et al., 2014; Cui et al., 2019; Thara et al., 1999; López-Ráez et al., 2010; Li et al., 2017; Harel et al., 2014). Interestingly, comparing B. cinerea induced gene expression with heat-shock induced gene expression in published datasets yields several defense related genes and transcription factors which are induced in both cases, and could be promising targets for future research (Supplemental Figure S3 ).