4 Discussion
Phosphorus and phytohormones play pivotal roles in regulating diverse developmental and physiological processes of plants (Raghothama & Karthikeyan, 1999; Vidhyasekaran, 2015). Pi starvation signaling crosstalks with hormone pathways to appropriately adapt Pi homeostasis in response to changing environmental conditions (Yuan & Dong, 2008; Baek et al. , 2017). Transcriptomic studies have revealed changes in the expression of hormone-responsive genes under Pi starvation inArabidopsis (Hammond et al. , 2003; Misson et al. , 2005; Bustos et al. , 2010; Woo et al. , 2012) and in rice (Wasaki et al. , 2006; Li et al. , 2010; Secco et al. , 2013). Pi starvation enhances plant sensitivity to auxin through overexpression of the auxin receptor TIR1 and the polar transport inhibitor BFA, with subsequent modification of the root system (López-Bucio et al. , 2000; Nacry et al. , 2005; Pérez-Torres et al. , 2008). Pi starvation also suppresses cytokinin levels via reducing expression of the cytokinin receptorCRE1 , thus decreasing plant sensitivity to cytokinin and root length (Martin et al. , 2000; Franco-Zorrilla et al. , 2002; López-Bucio et al. , 2002). Pi starvation additionally suppresses gene expression of enzymes involved in GA metabolism and increases accumulation of the negative regulator protein DELLA to decrease the level of bioactive GA (Jiang et al. , 2007). Pi starvation also regulates the transport, synthesis, and catabolism of abscisic acid during changes of the root system architecture (Jaschke et al. , 1997; Ciereszko & Kleczkowski, 2006). Notably, interaction between Pi starvation signaling and hormones is also involved in plant defense systems; for example, Pi starvation has recently been demonstrated to induce the JA pathway and enhance resistance to insect herbivory in dicot Arabidopsis (Khan et al. , 2016). Here, we demonstrated that adaptation to Pi starvation in monocot rice resulted in enhanced bacterial resistance through activation of the JA response (Figures. 1 and 2).
Specifically, we demonstrated that transcriptional regulation ofOsMYC2 by OsPHR2 was integral to Pi starvation-induced promotion of the JA signaling. In Pi starvation signaling, PHRs are known to regulate PSI genes via binding to P1BS elements (Rubio et al. , 2001; Zhou et al. , 2008; Bustos et al. , 2010).AtPHR1 partially controls Pi deficiency-triggered induction of JA signaling in Arabidopsis (Khan et al. , 2016), but the molecular mechanism remains to be elucidated. Here, we identified three P1BS cis -elements in the 2-kb promoter region of OsMYC2and further confirmed that OsPHR2, the homolog of AtPHR1 and thus the central regulator in rice (Wu et al. , 2013), was directly targeted to the promoter of OsMYC2 both in vivo andin vitro (Figure 3). Expression of OsMYC2 was enhanced inOsPHR2 overexpression mutants but suppressed in OsPHR2T-DNA insertion mutants grown under normal condition (Figure 4), further confirming expression of OsMYC2 as controlled by OsPHR2. Moreover, both the expression patterns of JA-synthesis genes and the basal MeJA level behaved in a similar manner as OsMYC2 inOsPHR2-Ov1 and phr2 mutants (Figures 4 and 5); this is consistent with the previous finding in Arabidopsis that MYC2/MYC3/MYC4 directly control the water spray-induced accumulation of JA (Van Moerkercke et al. , 2019). In this work, we additionally demonstrated that in myc2 mutants, neither the expression of JA-responsive genes nor MeJA production were altered in either the presence or absence of Pi (Figure 6), suggesting that activation of the JA signaling by Pi starvation depends on OsMYC2 . JA-IIe is the bioactive form in JA signaling, we also noticed the JA-IIe was not changed upon Pi starvation treatment or in OsPHR2 mutants (Figure S1). However, evidences have shown that JA signaling is clearly activated by repeated touching, wounding and oral secretion or short-term exposure to gaseous NO2 while JA-Ile levels remained unchanged in Arabidopsis (Chehab et al . 2012; Lange and Lange 2015; Bozorov et al., 2017; Mayer et al . 2018), supporting the notion that JA signaling can turn on without measurable increase in JA-Ile (Thierry et al ., 2019). Thus, we speculate that the transcriptional regulation of OsMYC2 by OsPHR2 reveals direct crosstalk between JA and Pi starvation signaling at the molecular level.
Prior studies have revealed that activation of JA signaling plays a positive role in Xoo resistance in rice (Koeduka et al. , 2005; Tao et al. , 2009; Deng et al. , 2012; Yamada et al. , 2012; Uji et al. , 2016; Ke et al. , 2020; Onohata & Gomi, 2020). For example, overexpressing OsWRKY45 andOsC3H12 enhanced Xoo resistance, accumulation of JA, and expression of JA signaling genes (Tao et al. , 2009; Deng et al. , 2012). Exogenous application of JA also enhanced resistance to bacterial blight, and this JA-induced Xoo resistance could be inhibited by overexpressing OsJAZ8∆C , which lacks the Jas domain (Yamada et al ., 2012) In the present work, we observed Pi starvation to enhance resistance to Xoo , accompanied by elevated expression of JA-responsive genes and accumulation of MeJA (Figures 1 and 2). In addition, we found that OsPHR2-Ov1 mutants (which have an activated JA signaling with unchanged JA-IIe level) were more resistant while phr2 mutants (which have a suppressed JA signaling) were more susceptible compared with the wild-type NIP plants (Figures 4, 5 and S1). Nevertheless, repetitive mechanical stimulation and NO2 fumigation enhances Arabidopsisresistance to Botrytis cinerea with activated JA signaling in JA-IIe steady-stage levels (Chehab et al . 2012; Mayer et al . 2018). Evidences also show that JA-IIe is not required to activate JA mediated systemic defenses to herbivory in Nicotiana attenuateand Solanum nigrum (Doorn et al., 2011; Bozorov et al., 2017). Therefore, we speculated that the JA signaling is involved in OsPHR2-mediated anti-Xoo defense. InArabidopsis , AtMYC2 and AtERF3 antagonistically repress JA-induced pathogen defense genes, and thus myc2-2mutants show increased sensitivity to the necrotrophic pathogenBotrytis cinerea (Lorenzo et al. , 2004; Zhai et al. , 2013). In tomato, however, MYC2 -silenced plants display enhanced resistance to Botrytis cinerea , as MYC2 and MTF ETHYLENE RESPONSE FACTOR.C3 synergistically and preferentially modulate pathogen-responsive genes (Du et al. , 2017). In rice, transgenic plants overexpressing OsMYC2 display a JA-hypersensitive phenotype and are more resistant to Xoo (Uji et al. , 2016); meanwhile, in OsMYC2 RNAi plants, the JA-inducible expression of many defense-related genes and JA-dependent activation of the biosynthetic pathways for specialized metabolites are both compromised (Ogawa et al. , 2017). Here, our data revealedOsMYC2 CRISPR/Cas9 mutants to exhibit a JA-insensitive phenotype and greater susceptibility to Xoo infection (Figure 6). In addition, the Xoosusceptibility of myc2 lines was not enhanced by Pi starvation (Figure 6), suggesting involvement of OsMYC2 in Pi starvation-mediated Xoo resistance. Considering OsPHR2 physically binds to the OsMYC2 promoter to regulate OsMYC2expression, resulting in consequent activation of the JA signaling (Figures. 3-5), we speculated that activation of the JA response resulting from the transcriptional regulation of OsMYC2 by OsPHR2 contributes, at least partially, to the bacterial defense induced by Pi starvation.
During its growth and development, rice is confronted by simultaneous nutrition deficiency and pathogen attack, such as Pi starvation (or low Pi) and bacterial blight. Here, we found a positive effect of Pi starvation on resistance to Xoo . Together with the well-known SPXs-PHR1 working model of the Pi starvation signaling pathway, we propose a model illustrating that Pi starvation- and JA- signaling function synergistically and positively to control rice resistance toXoo infection (Figure 7). When grown under the Pi-sufficient condition, OsSPXs interact with OsPHR2 with high binding affinity, prevent its binding to the P1BS motifs in the promoter of OsMYC2 , thus OsMYC2 is expressed at a solely basal level. Under the Pi-starvation condition, weakened interaction of OsSPXs-PHR2 allows PHR2 to up-regulate OsMYC2 , thereby enhancing expression ofOsMYC2 and consequently activating the JA response and JA-mediated antibacterial resistance. Our findings reveal a novel mechanism for crosstalk between Pi-starvation signaling and the JA pathway and its positive role in rice antibacterial immunity, and provide new insight into how plants adjust the balance between growth and defense by integrating nutrient supply and phytohormone signaling.