1 INTRODUCTION
In the natural environment, terrestrial plants are under relentless challenge by a great many biological organisms including viruses, bacteria, fungi, and oomycetes (Paulo Jose Pl Teixeira, 2019). Throughout a long evolutionary period, plants have acquired a suite of defense mechanisms including the ability to distinguish beneficial microbes from pathogens and to respond defensively when attacked by pathogens. Higher plants perceive microbial or host-derived immunogenic molecular patterns (MAMPs or DAMPs) and more variable pathogen effector proteins delivered to plant cells by PRRs (pattern recognition receptors) on the cell surface and intracellular immune receptors NLRs (nucleotide-binding, leucine-rich repeat receptors) (Ausubel, 2005; Bacete et al., 2018; Kourelis and Van der Hoorn, 2018; Van de Weyer et al., 2019). Triggering of plant surface PRRs and intracellular NLRs activates downstream signaling events, such as rapid phosphorylation of receptor-like cytoplasmic kinases (RLCKs), calcium inward flow, production of reactive oxygen species, activation of calcium-dependent kinases (CPKs), mitogen-activated protein kinase (MAPK) cascade reaction heterotrimeric G proteins, and production of phytohormones, activation of defense genes, phytoalexin induction. These signaling events contribute to plant resistance to pathogens (Bürger, 2019; Meng and Zhang, 2013; Qi et al., 2017; Seybold et al., 2014; Tang et al., 2017).
At the same time, plants are equipped with a range of induced defense mechanisms to withstand complex and harsh environments (Pieterse et al., 2014). The more established studies on induced resistance include systemic acquired resistance (SAR) and induced systemic resistance (ISR) (Conrath et al., 2002). In general, SAR is directly elicited by plant exposure to toxic, non-toxic and non-pathogenic microorganisms (Conrath et al., 2002), whereas ISR is triggered by some non-pathogenic rhizosphere microorganisms, such as plant growth-promoting bacteria (PGPR) (Van der Ent et al., 2008). ISR and SAR act through different signaling pathways. It was found that ISR induced by P. fluorescens WCS417r was blocked in the Arabidopsis JA signaling pathway mutant jar1, the ET signaling pathway mutant etr1, and the PR gene nonexpressing mutant npr1 (Pieterse et al., 1998), suggesting that P. fluorescens WCS417r-mediated ISR is dependent on the JA/ET signaling pathway and the NPR1 gene. The P. fluorescens WCS417r has been shown to trigger ISR in a variety of plants. It was shown that P. fluorescens WCS417r enhanced resistance to Fusarium oxysporum in the above-ground parts of the plant and produced more phytoalexin at the site of pathogen infection after root treated by P. fluorescens WCS417r (Van Wees et al., 1999). The results showed that PGPR strain P. fluorescens S97 triggered leaf ISR after root colonization in Leguminosae. The P. fluorescens WCS417r has been described to elicit ISR against a range of pathogens, including Xanthomonas campestris pv. campestrisand Pst DC3000 (Van der Ent et al., 2008). It was revealed that oxalic acid secreted byBacillus sp. resisted B. cinerea by activating the JA and ET signaling pathways in tomato (Yu et al., 2022). The plant growth-promoting rhizobacteria Bacillus amyloliquefaciens HK34 can trigger ISR against P. cactorum in Panax ginseng (Lee et al., 2015). There was a study showing that lncRNAs played an important role in biocontrol bacteria P. putidaSneb821 to induce tomato resistance against Meloidogyne incognitainfection (Yang et al., 2020). In addition to Pseudomonas , various strains of Bacillus , fungi, and viruses can also stimulate ISR, reducing the incidence and severity of crop diseases significantly. A wealth of studies has investigated the molecular mechanism of rhizosphere-triggered ISR. P. fluorescensWCS417r-ISR in Arabidopsis was shown to have no activation of PR genes in systemic leaf tissue (Pieterse et al., 1996) and be independent of SA (Pieterse et al., 2000). However, in opposition to the ISR triggered by P. fluorescens WCS417r, it was found that plant growth-promoting bacteria B. cereus AR156 triggered the ISR inArabidopsis thaliana by activating both SA and JA/ET signaling pathways in an NPR1-dependent manner, thereby enhancing plant resistance to Pst DC3000 (Niu et al., 2011). Further studies showed that WRKY11 and WRKY70 play essential roles in regulating the signaling pathway of B. cereus AR156-triggered ISR through activation of JA and SA signaling pathways, respectively (Jiang et al., 2015). In addition, recent studies have shown that B. cereus AR156 triggers ISR against Pst DC3000 by suppressing miR472 and activating CNLs-mediated basal immunity in Arabidopsis (Jiang et al., 2020).
Phytoalexin is a small-molecule secondary metabolite synthesizedde novo after the plant senses the invasion of pathogens. Camalexin (3-thiazol-2’-yl-indole) is a sulfur-containing indole alkaloid unique to cruciferous plants and the predominant phytoalexin in the model plant A. thaliana (Ahuja et al., 2012). It was first isolated from the leaves of Camelina sativa after infection byAlternaria brassica (Browne et al., 1991). After the discovery of phytoalexins from potatoes infected with blast molds, a large number of phytoalexins have been identified from various plants, such as camalexin, capsidiol, scopoletin, resveratrol and pisatin (Ahuja et al., 2012; Holland and O’Keefe, 2010; Pedras et al., 2011; Yang et al., 2009). The biosynthesis of camalexin originated from Tryptophan (Piasecka et al., 2015). Tryptophan is converted to indole-3-acetaldoxime (IAOx) by two cytochrome P450 enzymes, CYP79B2 and CYP79B3 (Glawischnig, 2006; Glawischnig et al., 2004). When plants are infected by pathogenic bacteria, two other cytochrome P450 enzymes, CYP71A12 and CYP71A13, are produced, which transform IAOx to indole-3-acetonitrile (IAN) (Mller et al., 2015; Nafisi et al., 2007). IAN is further activated and derivatized. Finally, GS-IAN is transformed into camalexin under the machining of γ-glutamylpeptidase and cytochrome P450 enzyme CYP71B15 (PAD3) (Böttcher et al., 2009; Mucha et al., 2019; Parisy et al., 2007; Schuhegger et al., 2006).
Camalexin production has been reported to be induced by many pathogens, such as P. syringae , Alternaria brassicicola , B. cinerea , P. brassicas , and Sclerotinia sclerotiorum . Camalexin is also an important immune response against the invasion of these pathogens (Jun Tsuji, 1992; Schlaeppi et al., 2010; Stotz et al., 2011; Thomma et al., 1999). The synthetic gene of camalexin is expressed at low levels in the absence of biotic or abiotic stresses and is highly induced once subjected to pathogen invasion (Nafisi et al., 2007; Schuhegger et al., 2006). WRKY33, known for being one of the pivotal transcription factors in the camalexin synthesis pathway, can be induced to be up-regulated by a variety of fungi, oomycetes, and bacteria (Mao et al., 2011; Qiu et al., 2008). WRKY33 can bind to the CYP71A13and PAD3 promoter regions to regulate its expression (Birkenbihl et al., 2017; Mao et al., 2011; Qiu et al., 2008). Mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs), activated during plant perception of pathogens, play a crucial role in the induction of camalexin synthesis. Acting upstream of WRKY33, MPK3/6 and CPK5/6 enhance WRKY33 activity by phosphorylating the N-terminal Ser residue and Thr-229 of WRKY33 protein, respectively, with the former increasing its transactivation activity and the latter enhancing its DNA binding activity (Mao et al., 2011; Zhou et al., 2020). The jasmonate and ethylene signaling pathways are also involved in the induction of camalexin synthesis upon pathogen infection. The transcription factor ETHYLENE RESPONSE FACTOR1 (ERF1) integrates the jasmonic acid and ethylene signaling pathways and induces camalexin by upregulating the expression of CYP71A13 and PAD3 . ERF1 and WRKY33 interact in the nucleus and they mediate the synthesis of camalexin by targeting the promoters of CYP71A13 and PAD3 , interdependently and cooperatively (Zhou et al., 2022).
However, the mechanism of how phytoalexin is induced by beneficial microorganisms against broad-spectrum pathogens remains elusive. In the present study, we found that AR156 could induce ISR against broad-spectrum pathogens, such as P. capsici , B. cinerea, Pst DC3000. RNA sequencing found that AR156-triggered ISR could induce the accumulation of phytoalexin such as camalexin synthesis and secretion-related genes. Deeply research demonstrated that AR156-induced ISR is impaired in wrky33 , probably because the induction of camalexin accumulation by AR156 is dependent on WRKY33.Meanwhile, AR156 induced up-regulated expression of PEN3 and PDR12. Nevertheless, PEN3 and PDR12 were jointly involved in AR156-triggered ISR against fungi and oomycetes, while PEN3 was involved in AR156-triggered ISR against Pst DC3000. Our study firstly raises that WRKY33 is involved in regulating the accumulation and secretion of phytoalexin induced by AR156 as a core factor. Besides, our study systematically elucidates the mechanism of PGPR-triggered ISR resistance to broad-spectrum pathogens and offers a theoretical basis for developing and applying new biopesticides.