bs_bs_banner MOLECULAR PLANT PATHOLOGY (2015) 16(2), 150–163 DOI: 10.1111/mpp.12168 Derivative of plant phenolic compound inhibits the type III secretion system of Dickeya dadantii via HrpX/HrpY two-component signal transduction and Rsm systems YAN LI1, WILLIAM HUTCHINS2, XIAOGANG WU2, CUIRONG LIANG3, CHENGFANG ZHANG3, XIAOCHEN YUAN2, DEVANSHI KHOKHANI2, XIN CHEN3, YIZHOU CHE2, QI WANG1,* AND CHING-HONG YANG2,* 1The MOA Key Laboratory of Plant Pathology, Department of Plant Pathology, College of Agronomy & Biotechnology, China Agricultural University, Beijing 100193, China 2Department of Biological Sciences, University of Wisconsin, Milwaukee, WI 53211, USA 3School of Pharmaceutical & Life Sciences, Changzhou University, Jiangsu 213164, China leads to strong selective pressure to develop resistance against SUMMARY antibiotics (Cegelski et al., 2008; Escaich, 2008; Rasko and The type III secretion system (T3SS) is a major virulence factor in Sperandio, 2010). In the face of increasing antibiotic resistance, many Gram-negative bacterial pathogens and represents a par- the targeting of bacterial virulence factors rather than bacterial ticularly appealing target for antimicrobial agents. Previous survivability provides a novel alternative approach for the devel- studies have shown that the plant phenolic compound p-coumaric opment of new antimicrobials, as virulence-specific therapeutics acid (PCA) plays a role in the inhibition of T3SS expression of the would offer a reduced selection pressure for antibiotic-resistant phytopathogen Dickeya dadantii 3937. This study screened a mutations (Escaich, 2008; Rasko and Sperandio, 2010).The type III series of derivatives of plant phenolic compounds and identified secretion system (T3SS) represents a particularly appealing target that trans-4-hydroxycinnamohydroxamic acid (TS103) has an for antimicrobial agents because it is a major virulence factor in eight-fold higher inhibitory potency than PCA on the T3SS of many Gram-negative plant and animal pathogens (Cornelis, 2006; D. dadantii. The effect of TS103 on regulatory components of the Tang et al., 2006; Waterman and Holden, 2003; Yang et al., 2002). T3SS was further elucidated. Our results suggest that TS103 inhib- The T3SS in phytobacteria, also known as the hypersensitive its HrpY phosphorylation and leads to reduced levels of hrpS and response and pathogenicity (Hrp) system, is a syringe needle-like hrpL transcripts. In addition, through a reduction in the RNA levels structure which is responsible for the secretion and translocation of the regulatory small RNA RsmB, TS103 also inhibits hrpL at the of effector proteins into the host cells, where the effector proteins post-transcriptional level via the rsmB-RsmA regulatory pathway. subvert or inhibit the host cell’s defences or facilitate pathogenic- Finally, TS103 inhibits hrpL transcription and mRNA stability, which ity (Alfano and Collmer, 1997; Galán and Collmer, 1999; Ghosh, leads to reduced expression of HrpL regulon genes, such as hrpA 2004; Grant et al., 2006; Hueck, 1998; Yang et al., 2005). and hrpN. To our knowledge, this is the first inhibitor to affect the Dickeya dadantii 3937 (formerly named Erwinia chrysanthemi), T3SS through both the transcriptional and post-transcriptional a member of the Enterobacteriaceae family, is a Gram-negative pathways in the soft-rot phytopathogen D. dadantii 3937. pathogen which causes soft rot, wilt and blight diseases on a wide range of plant species (Bauer et al., 1994). D. dadantii pos- Keywords: plant phenolic compound, Rsm system, T3SS sesses a T3SS, which is encoded by the hrp gene cluster and inhibitor, type III secretion system, two-component signal thought to be coordinately regulated by various host and environ- transduction system. mental factors (Nasser et al., 2005; Yang et al., 2002). Similar to many phytopathogens, the expression of the T3SS of D. dadantii 3937 is repressed in nutrient-rich media, but induced in the plant apoplast or in nutrient-deficient inducing medium, which is con- sidered to mimic plant apoplastic conditions (Galán and Collmer, INTRODUCTION 1999; Tang et al., 2006). The well-studied T3SS of D. dadantii is Antibiotic treatment is the most commonly used strategy to regulated by the master regulator HrpL, which is a member of the control pathogenic infections. However, most antibiotics kill bac- extracytoplasmic function (ECF) family of alternative sigma factors teria by inhibiting cellular processes essential for survival, which that up-regulate many hrp genes downstream of the T3SS regu- latory cascade, such as hrpA (encoding a structural protein of the *Correspondence: Email: [email protected] or [email protected] T3SS pilus), dspE (encoding a T3SS effector) and hrpN (encoding a 150 © 2014 BSPP AND JOHN WILEY & SONS LTD Phenolic compounds inhibit the T3SS of D. dadantii 151 Fig. 1 Regulatory network controlling the Dickeya dadantii type III secretion system (T3SS). The D. dadantii T3SS is regulated by the HrpX/HrpY-HrpS-HrpL and GacS/GacA-rsmB-RsmA-HrpL regulatory pathways. The two-component signal transduction system HrpX/HrpY activates hrpS, which encodes a σ54 enhancer. HrpS is required for the expression of the alternative sigma factor, hrpL. HrpL activates the expression of genes encoding the T3SS apparatus and its secreted substrates. RsmA is a small RNA-binding protein that acts by decreasing the half-life of hrpL mRNA. GacS/GacA up-regulates the expression of rsmB, which increases the mRNA level of hrpL by sequestering RsmA. From this study, we observed that TS103 altered the hrpA promoter activity through both the HrpX/HrpY-HrpS-HrpL and rsmB-RsmA-HrpL pathways. TS103 inhibits hrpL at the post-transcriptional level through a decrease in expression of rsmB. In addition, TS103 inhibits hrpS transcription through suppression of phosphorylation of HrpY and also post-transcriptionally inhibits rpoN. ⊥, negative control; →, positive control. IM: Inner Membrane; OM: Outer Membrane. harpin protein) (Fig. 1) (Chatterjee et al., 2002; Wei et al., 1992; IcII-like protein, has been reported to negatively control the tran- Yang et al., 2010; Yap et al., 2005). The expression of hrpL is scription of rsmB by binding within the transcribed region of the regulated at both the transcriptional and post-transcriptional rsmB gene in P. carotovorum (Miller et al., 2000). Polynucleotide levels. HrpX/HrpY-HrpS regulates hrpL at the transcriptional level. phosphorylase (PNPase) has been reported to decrease the A two-component signal transduction system (TCSTS) HrpX/HrpY, amount of functional rsmB transcripts in D. dadantii (Zeng et al., encoded by genes in the centre of the hrp gene cluster, positively 2010). regulates hrpS, which encodes a σ54 enhancer-binding protein In response to microbial attack, plants activate defence (Fig. 1) (Tang et al., 2006). HrpS interacts with the σ54 (RpoN)- responses which lead to the induction of a broad spectrum of containing RNA polymerase holoenzyme and initiates the tran- antimicrobial defences (Montesano et al., 2005; Van Loon, 2000). scription of hrpL (Fig. 1) (Chatterjee et al., 2002; Yap et al., 2005). These induced defences are regulated by a network of intercon- The regulator of secondary metabolites (Rsm) RsmA–RsmB pair necting signal transduction pathways and eventually lead to the regulates hrpL at the post-transcriptional level. RsmA, a small production of defence molecules, such as phenylpropanoids RNA-binding protein, promotes hrpL mRNA degradation (Fig. 1) (Dixon and Paiva, 1995; Feys and Parker, 2000; Hahlbrock and (Chatterjee et al., 2002; Cui et al., 1995). RsmB, an untranslated Scheel, 1989). Phenylpropanoids are a group of secondary regulatory small RNA, binds to the RsmA protein and neutralizes metabolites produced by plants from L-phenylalanine. Our previ- the activity of RsmA on hrpL mRNA degradation by forming an ous reports have shown that the plant phenolic compound inactive ribonucleoprotein complex with RsmA (Chatterjee et al., p-coumaric acid (PCA), an intermediate in phenylpropanoid 2002; Liu et al., 1998) (Fig. 1). The global regulatory TCSTS, GacS/ biosynthesis, plays a role in the inhibition of T3SS expression of GacA, up-regulates the transcription of the regulatory small RNA D. dadantii 3937 (Li et al., 2009).With the aid of structure–activity RsmB (Tang et al., 2006). In addition to GacS/GacA, other regula- relationship (SAR) studies, the para positioning of the hydroxyl tors have been identified that control the expression of rsmB group in the phenyl ring and the double bond in PCA have been in the soft-rot pathogens Pectobacterium and Dickeya. KdgR, an predicted previously to be essential for its inhibitory activity © 2014 BSPP AND JOHN WILEY & SONS LTD MOLECULAR PLANT PATHOLOGY (2015) 16(2), 150–163 152 Y. LI et al. (Li et al., 2009). As the regulatory mechanism of the T3SS of The hrpA gene encodes the T3SS pilus, which is required for the D. dadantii 3937 is well understood, to develop T3SS inhibitors translocation of effector proteins into plant cells and is located which are more potent than PCA, a series of derivatives of plant downstream in the T3SS regulatory pathway (Fig. 1). A reporter phenolic compounds were screened using D. dadantii 3937 as a plasmid, pPhrpA, which carries a hrpA-gfp transcriptional fusion, model organism. One derivative, TS103, which showed an eight- was used to measure the effects of the derivatives of plant phe- fold higher potency in the inhibition of the T3SS vs. PCA, was nolic compounds on hrpA expression (Table 1). The wild-type cells selected and the regulators responsible for the inhibition of T3SS containing pPhrpA were grown in T3SS-inducing medium (MM) gene expression by TS103 were further elucidated. Our results supplemented with each of the compounds at a concentration of showed that TS103 inhibits the T3SS through both the HrpX/HrpY 100 μM. Green fluorescent protein (GFP) intensity, which is a TCSTS and Rsm systems. measurement of hrpA promoter activity, was assayed by flow cytometry. Among the derivatives of the plant phenolic com- pounds screened, 13 compounds at a concentration of 100 μM RESULTS showed strong inhibition of T3SS gene expression of D.