NATURE | VOL 415 | 28 FEBRUARY 2002 | www.nature.com 977 articles MAP kinase signalling cascade in Arabidopsis innate immunity Tsuneaki Asai*§, Guillaume Tena*§, Joulia Plotnikova*, Matthew R. Willmann*, Wan-Ling Chiu*, Lourdes Gomez-Gomez², Thomas Boller³, Frederick M. Ausubel* & Jen Sheen* * Department of Genetics, Harvard Medical School, and Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA ² Instituto de Desarrollo Regional, Seccio Ân de Biotecnologõ Âa, Campus Universitario s/n, E-02071 Albacete, Spain ³ Friedrich Miescher-Institute, PO Box 2543, CH-4002 Basel, Switzerland § These authors contributed equally to the work ............................................................................................................................................................................................................................................................................ There is remarkable conservation in the recognition of pathogen-associated molecular patterns (PAMPs) by innate immune responses of plants, insects and mammals. We developed an Arabidopsis thaliana leaf cell system based on the induction of early-defence gene transcription by ¯agellin, a highly conserved component of bacterial ¯agella that functions as a PAMP in plants and mammals. Here we identify a complete plant MAP kinase cascade (MEKK1, MKK4/MKK5 and MPK3/MPK6) and WRKY22/ WRKY29 transcription factors that function downstream of the ¯agellin receptor FLS2, a leucine-rich-repeat (LRR) receptor kinase. Activation of this MAPK cascade confers resistance to both bacterial and fungal pathogens, suggesting that signalling events initiated by diverse pathogens converge into a conserved MAPK cascade. The innate immune system is the ®rst line of inducible defence against infectious disease. A key function of innate immunity is the detection of PAMPs produced by infectious agents but not by host cells 1±3 . In insects and mammals, the recognition of PAMPs is often mediated by Toll and Toll-likereceptors (TLRs), respectively, with extracellular LRRs 2,4 . In plants, LRR domains are also found in the products of disease resistance genes 5±8 , which are receptors impor- tant for innate immunity. Recent studies have identi®ed bacterial ¯agellin as a PAMP that is recognized by the innate immune system in diverse organisms including insects, mammals and plants 9±11 . In mammals, TLR5 mediates the innate immune responses to ¯agellin 11 . In Arabidopsis, the LRR receptor kinase FLS2 is required for ¯agellin signalling 12 . A 22-amino-acid peptide ¯g22, corresponding to the most conserved domain of eubacterial ¯agellin, binds to FLS2 and induces defence responses in leaves 10,12,13 . In contrast to animals, the molecular mechanisms underlying the responses to PAMPs are largely unknown in plants 5±7 . In mammals, different TLRs speci®cally recognize distinct PAMPs but activate common and conserved signal transduction pathways 2,4 . In plants, many different types of pathogens and pathogen-derived elicitors also trigger similar defence responses 7,14±17 . The best candidates for components of convergent signalling pathways in plants are the mitogen-activated protein kinases (MAPKs) 7,16±19 . Although several MAPKs involved in plant defence response have been identi®ed 14,20±25 , the identity of the upstream receptors, MAPK kinases (MAPKKs) and MAPKK kinases (MAPKKKs), and the downstream transcription factors remain mostly unclear. To elucidate MAPK signalling cascades in plant innate immune responses, we developed an Arabidopsis protoplast transient expres- sion system in which transcription of early defence genes is activated by ¯g22 and in which the role of MAPK cascade components could be systematically tested and assigned. Using this system, we have identi®ed a complete plant MAPK cascade and WRKY transcription factors acting downstream of the ¯agellin receptor FLS2. Our data also suggest that this signalling pathway functions in response to both fungal and bacterial pathogens and potentially could be engineered to enhance the disease resistance of crop plants to a wide range of pathogens. Early defence gene transcription To dissect the early signal transduction pathways in plant innate immune responses, we ®rst established a leaf cell assay based on ¯g22 inducible transcription of early response genes in Arabidopsis mesophyll protoplasts 26 . Few reporter genes have been developed for the dissection of the early stages of defence signalling pathways in plants 27±29 . To identify genes that are induced by ¯g22-activated defence signalling, we created a subtracted complementary DNA library that represented messenger RNA species induced at various times by the elicitor ¯g22 in Arabidopsis mesophyll protoplasts 30 . Using ¯g22 rather than a pathogen or natural elicitors avoided the possibility that multiple elicitors could be functioning in parallel 12 . Furthermore, synchronous elicitation by ¯g22 is achieved more reproducibly in homogeneous mesophyll protoplasts than in intact leaves 26 . In the library we found well described defence genes, such as PAL1 (At2g37040 in the Arabidopsis Genome Initiative nomen- clature), GST1 (Atlg02930), PR1 (At2g19990) and PR5 (Atlg75040), which are induced by a variety of pathogens and elicitors at different stages of the defence response in many plant species 28,29,31 . These genes were also induced in Arabidopsis leaves in®ltrated with ¯g22 (not shown), suggesting that similar defence responses are induced by ¯g22 in isolated leaf protoplasts and in leaves of intact plants. To develop new reporter genes that are expressed early in the primary defence response, we focused on ¯g22-activated genes identi®ed in the subtraction library that are expressed at early time points (see below) and code for putative regulatory factors, including WRKY29 (At4g23550) 32 and FRK1 (FLG22-INDUCED RECEPTOR-LIKE KINASE 1; At2g19190), which encode a WRKY transcription factor (with a conserved WRKY DNA-binding domain) and a LRR receptor kinase, respectively. Induction of WRKY transcription factors and LRR receptor kinase genes by pathogens, pathogen-derived elicitors or salicylic acid has been demonstrated in the leaves of several plant species including Arabidopsis 28,29,32±34 . These results indicate that Arabidopsis proto- plasts treated with ¯g22 increase transcription of defence-related genes that are also expressed in intact plants after pathogen infection. Polymerase chain reaction after reverse transcription of RNA (RT-PCR) analysis showed that WRKY29, FRK1 and GST1 mRNA levels were increased in Arabidopsis protoplasts within 30 min after ¯g22 treatment (Fig. 1a), whereas induction of the extensively studied PR1 and PR5 genes occurred much later (Fig. 1a). Two stress-regulated genes, H 2 O 2 -inducible GST6 (At2g47730) and ABA-, cold-, or drought-responsive RD29A (AT5g52310) 35 , were © 2002 Macmillan Magazines Ltd