Innate immunity in plants Jonathan Cohn*, Guido Sessa* and Gregory B Martin*

55 Innate immunity in plants Jonathan Cohn*, Guido Sessa* and Gregory B Martin* Studies of receptors and signal-transduction components that play a role in plant disease resistance have revealed remarkable similarities with innate immunity pathways in insects and mammals. In plants, specific receptors encoded by disease-resistance genes interact with products of microbial effector genes to activate defence responses. Resistance proteins have been found to have motifs in common with components of immune response pathways in mammals and invertebrates, and to rely on similar downstream signalling components. In the future, the sharing of ideas among plant and animal biologists is likely to broaden our understanding of defence responses in diverse organisms. Addresses *Boyce Thompson Institute for Plant Research and Department of Plant Pathology, Cornell University, Tower Road, Ithaca, New York, NY 14853, USA Correspondence: Gregory B Martin; e-mail: gbm7@cornell.edu Current Opinion in Immunology 2001, 13:55 62 0952-7915/01/$ see front matter 2001 Elsevier Science Ltd. All rights reserved. Abbreviations avr avirulence CDPK calcium-dependent PK DD death domain HR hypersensitive response IL-1R IL-1 receptor IRAK IL-1R-associated kinase LRR leucine-rich repeat NBS nucleotide-binding site NO nitric oxide PK protein kinase PR pathogenesis-related R resistance RLK receptor-like kinase ROS reactive oxygen species SA salicylic acid SAR systemic-acquired resistance TIR Toll/IL-1R TMV tobacco mosaic virus Introduction Plants have evolved an array of rapid and efficient defence responses against a wide variety of pathogens including bacteria, fungi, viruses and nematodes. One of the most powerful weapons in a plant s arsenal against pathogen attack is the hypersensitive response (HR). The HR is characterised by rapid, localised cell death at the site of infection [1]. This cell death response likely benefits the plant by depriving pathogens of access to further nutrient sources and limiting pathogen proliferation. Additionally, there is evidence that suggests that signal molecules produced from dying cells are involved in the induction of a variety of defence-related genes [1,2]. A wide range of physiological changes are known to occur in response to pathogen attack, including the production of reactive oxygen species (ROS), transient ion-flux leading to intracellular ph changes, cell wall strengthening near the infection site, release of secondary signal molecules such as nitric oxide (NO) and the synthesis of antimicrobial products including phytoalexins and pathogenesis-related (PR) proteins. The PR proteins, for example glucanases and chitinases, are known to have both antifungal and antibacterial properties. The additive effects of these cellular changes result in the ability of a plant to protect itself from constant attack by microbial pathogens. Many responses involved in plant disease resistance are dependent upon interaction of pathogenic effector molecules with specific plant resistance (R) proteins [3]. Although the signalling pathways initiated by these interactions are just beginning to be unravelled [4], the past few years have seen dramatic advances in our understanding of the molecular principles of plant disease resistance. It has become clear that some of the molecular mechanisms involved in innate immunity in mammalian and insect systems are remarkably similar to the molecular mechanisms underlying plant disease-resistance responses [5 ]. It has been proposed, therefore, that innate immunity might be an evolutionarily ancient system of host defence [6]. In this review we discuss this possibility and the recent major advances in understanding the mechanisms by which plants defend themselves against pathogen attack. Mechanisms of the plant immune response Associated with the HR is the production of ROS, which is typically one of the earliest responses of plants to microbial pathogens. ROS generation in plants bears similarities to the oxidative burst described in mammalian neutrophils [7,8], which employs a NADPH-oxidase dependent system. Although plant homologs of two NADPH-oxidase components, gp91 phox and Rac, have been cloned recently, a role for these proteins in the plant oxidative burst has not yet been demonstrated [9,10]. Production of ROS, such as H 2 O 2 and superoxide ( O 2 ) radicals, results in cellular damage to both host and pathogen. Additionally, H 2 O 2 is likely to contribute to cell wall reinforcement, as it is known to be essential to cell wall lignification [1,11 ]. Cell wall strengthening around the site of pathogen ingress might limit microbial proliferation. ROS may also be critical to defence signalling. Indeed, ROS have been shown to induce a variety of defence genes in plants and contribute to increased resistance to Pseudomonas syringae pv. syringae in transgenic tobacco [12,13]. In plants, prior pathogen infection can result in the development of a heightened, systemic resistance to a secondary attack by a broad spectrum of pathogens. This immunity is referred to as systemic-acquired resistance (SAR; [14 ]). SAR is characterised by the induction of a number of

56 Innate immunity defence-related proteins, including many PR proteins. A large amount of evidence supports a role for salicylic acid (SA) in both SAR and disease resistance. Among the most compelling support for a role for SA comes from experiments with transgenic plants that express the bacterial nahg gene that encodes an enzyme, salicylate hydroxylase, which inactivates SA. Normally, plants responding to pathogen attack accumulate significantly higher levels of SA than uninfected plants. In several studies, transgenic plants expressing nahg did not accumulate SA after exposure to pathogens and were more susceptible to both pathogens that normally induce a resistance response and to pathogens that normally cause disease. Moreover, these plants failed to develop SAR or express PR genes in uninoculated leaves. This indicates that SA plays an important role required in SAR ([14 ] and references therein). Jasmonic acid and ethylene have been shown to induce the expression of antimicrobial peptides that are not induced by SA. Previous studies established that jasmonic acid and ethylene are involved in plant response to wounding and insect attack, which includes the activation of proteinase inhibitors [15]. Interestingly, jasmonic acid and ethylene apparently act antagonistically with the SA pathway, suggesting that initiation or control of multiple pathways is necessary for maintenance of disease resistance ([15] and references within). SAR requires the systemic movement of an as-yet uncharacterised signal molecule from infected tissue to distally located uninfected tissues. Although SA itself was initially believed to be the long-distance signalling molecule, this has not been demonstrated [16]. In fact, it has been suggested that NO might fulfill this role [17]. NO is a well-established secondary signalling molecule in mammalian systems and it was recently demonstrated that it plays a similar role in plant defence responses [17,18]. NO is not only capable of triggering expression of PR proteins but also necessary for induction of ROS-dependent cell death, acting synergistically with both ROS and SA [18,19]. The role of NO in cell death is not well understood but it has been suggested that redox signalling through NO and ROS is enhanced by SA in a feedback loop mechanism. Interestingly, a recent report suggested that ROS might act as long-distance signals that mediate SAR [20]. In this study, Arabidopsis plants inoculated with P. syringae displayed a rapid induction of secondary oxidative bursts in distally located uninoculated leaves, which were reported to lead to systemic micro-hrs. These secondary microbursts were dependent upon a primary oxidative burst at the site of infection. The most significant change in protein production associated with the HR and SAR is the accumulation of PR proteins. A wide variety of PR proteins have been characterised and are classified, mostly on sequence comparisons, into at least 11 different families [21]. Although the function of many PR proteins is not clear, several have been shown to have deleterious effects on pathogens in vitro. Indeed, a number of PR proteins have been shown to be glucanases or chitinases that damage fungal cell walls [21,22]. Experiments employing the co-expression of multiple transgenes in plants suggested that PR proteins are likely to act synergistically in planta [23]. The antimicrobial action of PR proteins might also indirectly contribute to overall plant immunity. For example, chitinase action on fungal cell walls is known to release breakdown products that induce the production of phytoalexins (low molecular weight, lipophilic antimicrobial compounds) and SAR [24]. Interestingly, plant chitinases were recently proposed to be molecular targets of selection in the co-evolution of plants and pathogens [25 ]. This is significant because most studies of plant pathogen co-evolution have focused on putative plant-receptor proteins rather than downstream PR proteins. Another important class of proteins induced during the defence response is the defensins, which also have been shown to be important proteins in animal disease resistance (see the review by Thomma et al., this issue, pp 63 68). Plant disease resistance: gene-for-gene interactions In many cases, the HR is initiated by a gene-for-gene interaction that involves a dominant R gene in the plant and a corresponding avirulence (avr) gene in the pathogen [26]. If either the pathogen or the host lacks the corresponding avr or R gene, then the plant microbe interaction results in disease. Pathogen-derived avr gene products are delivered to intercellular spaces or directly inside plant cells, where they interact with the products of plant R genes. The R proteins are either transmembrane or intracellular proteins that are presumed to initiate signaltransduction cascades upon ligand binding. Many R gene products share structural motifs, indicating that similar pathways might control resistance to diverse pathogens. To date, over 20 R genes have been identified and 5 classes are recognised: intracellular proteins with a nucleotidebinding site (NBS), a leucine-zipper motif and a leucine-rich repeat (LRR) domain; intracellular NBS LRR proteins with a region of similarity to the cytoplasmic domain of mammalian IL-1 receptor (IL-1R) and the Drosophila Toll proteins (i.e. the TIR [Toll/IL-1R] domain); intracellular protein kinases (PKs); proteins with an LRR domain that encodes membrane-bound extracellular proteins; and receptor-like kinases (RLKs) with an extracellular LRR domain. Each of these categories are detailed below. Many of the characterised R genes encode cytoplasmic proteins with regions believed to be involved in signal transduction. For example, the recently cloned Rx protein of potato, which confers resistance to the potato virus X, is a cytoplasmic protein that has predicted NBS regions and distinct LRRs [27]. The presence of an NBS region, also present in several ATP- and GTP-binding proteins, suggests that these proteins may play a role in the activation of a kinase or as a G protein [4]. The leucine-zipper region, usually present in R proteins of this class, is likely to be

Innate immunity in plants Cohn, Sessa and Martin 57 involved in protein protein interactions [4]. LRRs have been implicated in protein protein interactions and ligand binding in a wide variety of proteins [4]. It is generally believed that the LRR region present in many R genes is a major factor responsible for the specificity of pathogen recognition [4]. Consistent with this hypothesis is a recent report that indicated distinct alleles of the flax R gene, L, differed only in their LRR region [28 ]. The different alleles of the L locus of flax are known to confer specific resistance to distinct strains of Melampsora lini (flax rust). These data suggested that gene-for-gene specificity might be partially controlled by variability in the LRR regions of different R genes; however, in the case of one R protein from Arabidopsis (RPS5), the LRR region appears to be required for signalling events subsequent to pathogen perception [29]. Two inactive alleles of the RPS5 gene which mediates resistance to Pseudomonas syringae strains harbouring the avr gene, avrpphb were analysed and found to have mutations in their respective LRR regions. One of the RPS5 mutations affected the function of several other R genes, suggesting that at least one region of the LRR interacts with signalling components utilised by multiple R-gene products. As mentioned, not all R genes possess LRR regions and it is, therefore, very likely that there are other factors controlling gene-for-gene specificity. Indeed, it was recently shown that differences in the non-lrr regions of LRR-type R genes play a role in specificity determination [30]. One class of R genes includes putative cytoplasmic proteins that share a TIR domain [3]. An example of this type of R protein is the well-characterised N protein of tobacco, which confers resistance to tobacco mosaic virus (TMV) [31]. The N gene encodes two transcripts that are both required to confer complete resistance to TMV [32]. Although other R genes have been shown to encode two or more transcripts, it is not clear if multiple transcripts are necessary for disease resistance. A distinct class of cytoplasmic R proteins has been identified in tomato. The product of the first gene-for-gene type of R gene cloned (Pto) confers resistance in tomato to P. syringae strains carrying the avrpto gene [33]. Pto is a member of a small family of serine/threonine PKs, which also includes the kinase, Fen, that confers sensitivity to the herbicide, fenthion [34]. These proteins contain the 11 subdomains common to all PKs and have a myristylation motif at the amino terminus. Myristylation is known to play a role in subcellular localisation of proteins, but experiments examining a role for this motif in Pto indicate that it is not required for disease resistance [35]. Pto and Fen both require another protein, Prf, for activity [36]. Interestingly, Prf contains a region of LRRs and a NBS similar to the first class of R proteins described above. Several R genes have been isolated that encode proteins with extracytoplasmic domains. For example, the Cf proteins of tomato, which confer resistance to strains of the leaf mould Cladosporium fulvum carrying the appropriate avr genes, are glycoproteins with extracytoplasmic regions of LRRs attached to a transmembrane region and a small cytoplasmic tail [37 39]. A second class of R proteins with an extracytoplasmic domain is typified by the Xa21 protein of rice, which confers resistance to Xanthomonas oryzae pv. oryzae. This protein has a LRR domain, a single transmembrane region and an intracellular serine/threonine kinase domain [40]. Xa21 is a member of the growing class of plant RLKs that appear to have very diverse roles in plants [41]. A recent report suggested that RLKs share a general signalling mechanism in that ligand interaction results in activation of the kinase domain [42 ]. In this study, the extracellular LRR region and transmembrane domain of the Arabidopsis BRl1 RLK was fused to the intracellular serine/threonine kinase domain of Xa21. BRl1 is implicated in brassinosteroid signalling in Arabidopsis. The fusion construct, transfected into rice cells, was able to elicit defence responses upon stimulation with brassinosteroids. This study suggests that the LRR region, at least when present in the extracellular region of R genes, is important for ligand binding. Intriguingly, this study also suggests that the kinase domain of RLK proteins is responsible for initiation of highly specific downstream signalling pathways. Type III secretion of avirulence proteins and intracellular recognition by resistance proteins As mentioned above, many R genes encode cytoplasmic proteins, suggesting that pathogen-produced effector proteins (i.e. the products of the respective avr genes) are active inside the cell. The avirulent phenotypes conferred by bacterial avr genes are dependent upon the functional expression of the pathogen HR and pathogenicity (hrp) genes, which encode proteins of the type III secretion pathway [43 ]. This system was originally characterised in bacterial pathogens of animals, such as Yersinia and Salmonella species, which inject pathogenic effector-proteins into host cells through the type III system. Several Avr proteins are now known to be secreted by the type III secretion pathway [43 ] and the localisation of Avr proteins in the plant cell has become an area of intense research. There is ample evidence to suggest that many Avr proteins interact with R proteins inside the plant cell. The first evidence of this interaction came from two separate studies [44,45] of the action of the P. syringae avrpto gene in plant cells. Transient expression of avrpto in plant cells induced a Pto-dependant HR. Moreover, AvrPto and Pto were shown to interact in a yeast two-hybrid system. A similar interaction was demonstrated for the Avr-Pita protein from the rice blast fungus, Magnaporthe grisea, and the product of the rice R gene, Pi-ta [46 ]. In this study, protein protein interaction was demonstrated both by using the yeast-two hybrid system and by using an in vitro binding assay. A recent study further supports the intracellular interaction

58 Innate immunity Figure 1 Signalling pathways leading to activation of defense response genes in mammals, insects and plants share common components. For example, related PKs occur in the IL-1R, Toll and Pto Prf defence pathways in humans, Drosophila and tomato, respectively. Other common elements in defence pathways include LRRs, DDs and the TIR domain. Several R proteins in plants also contain putative NBSs. (a) In humans, IL-1 binds IL-1R (which has a TIR); the adaptor MyD88 (which has a DD) then links this to IRAK (a PK); this releases NF-κB from its inhibitor (I-κB); NF-κB then activates transcription of genes important in host defence. (b) A similar sequence occurs when Spätzle binds Toll: eventually Dorsal is released from Cactus; in addition, subsequent Toll-mediated resistance to disease requires the transcription factors, Dif and Relish. (c) Tomato Pto-mediated signalling may involve complexes similar to those found in IL-1R- and Toll-mediated signalling: Pto (which requires Prf for its activity) is homologous to IRAK and Pelle. Pto interacts with Pti1, which is involved in the plant HR; Pto also interacts with Pti4/5/6, which are transcription factors that may be involved in regulation of PR genes. (d) Other plant proteins that may also signal in a similar fashion to IL-1R and Toll are tomato Cf-9 and rice Xa21: Cf-9 and Xa21 have (a) IL-1R Cell surface TIR MyD88 DD PK IRAK NF-κB (b) Toll LRR TIR Tube DD PK Pelle Dorsal/Dif/Relish extracytoplasmic domains and ligand interaction may result in kinase activity; presumably, avr9 interaction with Cf-9 activates a CDPK that may be involved in (e) N TIR NBS LRRLRR Immune response genes (d) Xa21 Cf-9 LRR PK LRR PK Pto Pti4/5/6 (c)? Prf NBS NBS LRR Current Opinion in Immunology host defence responses. (e) The tobacco N protein contains a TIR domain, an NBS and a LRR and is involved in resistance to TMV. of R proteins and Avr proteins in vivo [47 ]. In this work, the authors used a transient assay to express the RPS2 and AvrRpt2 proteins in leaf mesophyll protoplasts and showed that the two proteins co-immunoprecipitated along with at least one additional 75 kda protein. This is the first report clearly demonstrating a physical interaction of R proteins and Avr proteins in vivo. Some Avr proteins possess eukaryotic-nuclear-localisation-like sequences and interact with host nuclear factors, which might affect host defencegene transcription [48]. It has been observed that several Avr proteins have amino-terminal myristylation motifs; however, a function for this type of domain was not clear until recently. Two recent reports show that myristylation and acylation of Avrs inside the plant cell mediates their translocation to the plasma membrane, which enhances their functionality [49,50 ]. Function of resistance proteins in plant defence signalling: similarities to innate immunity in animals The observation that some R gene products share similar amino acid motifs with the TIR family of proteins is likely to provide insights into how these proteins interact with downstream signalling components involved in disease resistance (Figure 1). IL-1R plays a critical role in immunity and inflammation responses of mammals by initiating a signalling cascade upon binding its cognate ligand, the cytokine IL-1, which results in activation of the transcription factor NF-κB [51 ]. Binding of IL-1 stimulates recruitment of a number of molecules that form a protein complex. Upon ligand binding, the adaptor molecule MyD88 binds to the receptor and interacts through a conserved death domain (DD; so called because it was originally defined in proteins involved in apoptosis) with the PK, IRAK (IL-1Rassociated kinase) [51 ]. Upon interaction with the IL-1R complex, IRAK is phosphorylated and recruits the adaptor protein TRAF-6, which subsequently interacts with NIK (NF-κB-inducing kinase), resulting in phosphorylation of IKK (I-κB kinase). Subsequently, IKK phosphorylates I- κb (an inhibitor of NF-κB), targeting it for degradation through the ubiquitin proteasome pathway. Degradation of Iκ-B results in dissociation from NF-κB, which is then free to translocate to the nucleus and activate gene transcription. Several recent reviews have elaborated further on IL-1 signalling pathways (for example, see [51 ]). A pathway with similar components has been appreciated for some time in Drosophila (Figure 1). The Toll pathway controls both dorsoventral patterning and production of antimicrobial signals (see the review by Krutzik, Sieling and Modlin, this issue, pp 104 108). Upon binding a proteolytically cleaved form of its ligand, Spätzle, Toll initiates a signalling cascade that requires the proteins Tube and Pelle (analogous to MyD88 and IRAK, respectively), resulting in the degradation of Cactus, a Drosophila I-κB homolog, which is complexed with Dorsal, a transcription

Innate immunity in plants Cohn, Sessa and Martin 59 factor related to the Rel/NF-κB family of proteins. Tollmediated activation of antifungal and antibacterial peptide production requires the activation of two distinct Rel-type transcription factors, Relish and Dif. The resistance protein Pto of tomato bears remarkable similarity to components of the signal transduction pathways of animals that control the innate immune response. Although it possesses no TIR domain, Pto is homologous to the proteins IRAK and Pelle of mammals and Drosophila, respectively. It was recently discovered that Pto autophosphorylates in vitro through an intramolecular mechanism on serine and threonine residues [52]. Importantly, the ability of Pto to autophosphorylate was found to be necessary for interaction of Pto with AvrPto [53 ]. Mutation of the major site of Pto phosphorylation, Thr38, abolished receptor ligand interaction and development of an HR. Notably, this critical residue is conserved in both IRAK and Pelle. Domain swapping between Pto and Fen identified a region of Pto that is necessary for both AvrPto interaction and development of the HR [54]. Interestingly, a threonine residue in this region, Thr204, which is necessary for AvrPto Pto interaction, corresponds to a conserved threonine residue in both IRAK and Pelle. How can physical interactions of Avr proteins with the products of R genes result in the activation of downstream signalling components? One possible mechanism is that Avr R interaction results in a conformational change in the activation domains of critical enzymatic components (e.g. kinases). In support of this hypothesis, a Pto mutant (Tyr207 Asp), containing a substitution in the activation domain, was able to elicit a HR in the absence of AvrPto [55]. It should be noted that this mutant still required the presence of active Prf. Additionally, a mutation in the Pto autophosphorylation site, Ser198, interferes with the elicitation of a HR but not with the interaction with AvrPto [53 ]. It is possible that AvrPto binding to Pto causes a conformational change in Pto, resulting in its activation and subsequent phosphorylation of downstream substrates. Autophosphorylation also may be required for the interaction of Pto with downstream signalling components. The latter hypothesis is supported by the finding that a mutation of Ser198, the autophosphorylation site, perturbs interaction of Pto with another serine threonine kinase, Pti1, and disrupts interaction with two other proteins that remain to be functionally characterised: Pti3 and Pti10 [53 ]. Pti1 is a cytoplasmic protein that was recently shown to interact with Pto in a yeast two-hybrid screen [56]. Pti1 enhanced the HR when expressed in tobacco leaves inoculated with P. syringae pv. tabaci expressing the avrpto gene. Pti1 has been shown to autophosphorylate in vitro, in addition to being specifically phosphorylated by Pto in vitro [52,56]. As in mammalian systems, studies of in vivo phosphorylation using specific substrates and enzymatic inhibitors in plants have shown that PKs and phosphatases are critical for activation of defence responses [4]. For example, it was demonstrated that MAP kinases were activated in plants in response to pathogen inoculation [57,58]. MAP-kinase signalling in mammalian systems and yeast is known to be one of the major pathways by which extracellular signals are transduced. Romeis et al. [59 ] reported the identification of a calcium-dependent PK (CDPK), a member of a class of serine/threonine kinases that is unique to plants (and some protists), which is specifically activated upon recognition of the Avr9 protein from C. fulvum by the Cf-9 resistance protein from tomato. In addition, phosphorylation-dependent activation of the CDPK was accompanied by an increase in enzymatic activity. CDPKs are believed to be analogous to PKC isomers characterised in animal systems. Interestingly, PKC activity is required for induction of the defence activated oxidative burst in macrophages. The fact that Pto-mediated defence signalling requires Prf suggests that the Pto signalling cascades might require recruitment of a signalling complex of several proteins, similar to signalling complexes involved in IL-1R- and Toll-mediated signalling pathways. Consistent with this hypothesis is the fact that Prf contains a region of LRRs, which have been shown to mediate protein protein interactions. As mentioned above, Pto has been shown to interact with other proteins, such as Pti1, using a yeast twohybrid system. In addition to Pti1, Pto was shown to interact with Pti4, Pti5 and Pti6 [60], which are putative transcription factors. Pto autophosphorylation was required for physical interaction of Pto with Pti4, 5 and 6, suggesting that these proteins might be part of a signalling complex recruited by Pto phosphorylation. Transcriptional activation of plant defencerelated genes Although believed to be important players in disease resistance, relatively little is known about the transcriptional regulation of defence-related PR genes. One mechanism by which these genes may be activated is through specific regulation of transcription factors such as Pti4/5/6. In gelshift assays, Pti4/5/6 bind to a cis-acting element (the GCC box) required for ethylene responsiveness present in promoters of many PR genes [60,61 ]. In addition, mrna transcript levels of Pti4 and Pti5 increased in response to infection by P. syringae [62]. Interestingly, gel-shift analysis indicated that Pti4 binding to a GCC box element was enhanced by phosphorylation of Pti4 by Pto [61 ]. The NPR1 protein of Arabidopsis (also known as NIM1) is known to play a critical role in SAR. NPR1 is believed to be involved in SA-mediated signalling pathways that lead to transcriptional activation of PR proteins. NPR1 shares some similarities with the mammalian transcriptional regulator Iκ-B, and possesses an ankyrin-like-repeat domain known to mediate protein protein interaction [63,64]. In three recent studies using the yeast two-hybrid system, NPR1 was shown to interact directly with members of the

60 Innate immunity TGA family of transcription factors in Arabidopsis [65 67 ]. The TGA proteins are members of the bzip family of transcription factors and are known to recognise elements in the promoters of defence genes in plants. Each of the three studies showed that NPR1 was able to enhance the ability of TGA factors to bind to promoter elements present in PR genes. The results of these studies suggest that NPR1 is directly involved in transcriptional activation of PR genes through interaction with TGA transcription factors. Owing to the fact that NPR1 shares similarities to I-κB, it has been proposed that NPR1 might act as a negative regulator of gene transcription, similar to mammalian I-κB [66 ]. This mechanism of NPR1 action, however, has not been demonstrated. In fact, most of the data available for NPR1 action suggest that it acts as a positive regulator of defence responses. Conclusions Our understanding of the immune response of plants to pathogen attack has been greatly enhanced by the recent identification of R genes and biochemical analysis of their products. It is very intriguing that so few distinct classes of R genes have been identified. This suggests that plants have evolved a limited number of mechanisms to defend themselves against the plethora of pathogens that they encounter. A better understanding of how the interaction of R gene products and specific Avr factors activates defence pathways should allow for the improvement of disease resistance in economically important crops. Comparison of the components of the signalling pathways used in plant defence and in mammalian innate immunity has provided intriguing clues into how R genes might regulate resistance to pathogenic microbes. Future concerted efforts of researchers studying plant and animal defence signalling pathways should provide for a greater understanding of innate immunity in a wide variety of multicellular organisms. Ackowledgements Work in our laboratory on the Pto pathway is supported by the National Science Foundation (MCB-9896308), the United States Department of Agriculture (NRI-99-35301-7973) and the David and Lucile Packard Foundation. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. Hammond-Kosack KE, Jones JDG: Resistance gene-dependent plant defense responses. Plant Cell 1996, 8:1773-1791. 2. Dangl JL, Dietrich RA, Richberg MH: Death don t have no mercy: cell death programs in plant-microbe interactions. Plant Cell 1996, 8:1793-1807. 3. Hammonnd-Kosack K, Jones JDG: Plant disease resistance genes. Annu Rev Plant Physiol Plant Mol Biol 1997, 48:575-607. 4. Martin GB: Functional analysis of plant disease resistance genes and their downstream effectors. Curr Opin Plant Biol 1999, 2:273-279. 5. Hoffmann JA, Kafatos FC, Janeway CA, Ezekowitz RAB: Phylogenetic perspectives in innate immunity. Science 1999, 284:1313-1318. A comparison of the molecular mechanisms thought to contribute to innate immunity in Drosophila and mammals. The article emphasises the marked conservation of innate defences in mammals and insects, and suggests a common ancestry of these systems. 6. Medzhitov R, Janeway CA: An ancient system of host defense. Curr Opin Immunol 1998, 10:12-15. 7. Dwyer SC, Legendre L, Heinstein PF, Low PS, Leto TL: Plant and human neutrophil oxidative burst complexes contain immunologically related proteins. Biochim Biophys Acta 1996, 1289:231-237. 8. Groom QJ, Torres MA, Fordham-Skelton AP, Hammond-Kosack KE, Robinson NJ, Jones JDG: rboha, a rice homologue of the mammalian gp91phox respiratory burst oxidase gene. Plant J 1996, 10:515-522. 9. Keller T, Damude HG, Werner D, Doerner P, Dixon RA, Lamb C: A plant homolog of the neutrophil NADPH oxidase gp91phox subunit gene encodes a plasma membrane protein with Ca 2+ binding motifs. Plant Cell 1998, 10:255-266. 10. Kawasaki T, Henmi K, Ono E, Hatakeyama S, Iwano M, Satoh H, Shimamoto K: The small GTP-binding protein rac is a regulator of cell death in plants. Proc Natl Acad Sci USA 1999, 96:10922-10926. 11. Bolwell GP: Role of active oxygen species and NO in plant defence responses. Curr Opin Plant Biol 1999, 2:287-294. Nitric oxide (NO) is a well-established second messenger molecule in animal systems but has only recently been studied as a signal molecule in plants. This review highlights the current knowledge of NO signalling in plant defence pathways and also provides an outline of the role that reactive oxygen species (ROS) play in plant defence. 12. Lamb C, Dixon RA: The oxidative burst in plant disease resistance. Annu Rev Plant Physiol Plant Mol Biol 1997, 48:251-275. 13. Chamnongpol S, Willekens H, Moeder W, Langerbartels C, Sandermann HJ, Montagu MV, Inze D, Camp WV: Defense activation and enhanced pathogen tolerance induced by H 2 O 2 in transgenic tobacco. Proc Natl Acad Sci USA 1998, 95:5818-5823. 14. Dempsey D, Shah J, Klessig DF: Salicylic acid and disease resistance in plants. Crit Rev Plant Sci 1999, 18:547-575. A thorough overview of the current understanding of the role that SA plays in plant defence responses with reference to signal-transduction mechanisms and a brief overview of SA-independent signalling mechanisms. 15. Dong X: SA, JA, ethylene, and disease resistance in plants. Curr Opin Plant Biol 1998, 1:316-323. 16. Rasmussen JB, Hammerschmidt R, Zook MN: Systemic induction of salicylic acid accumulation in cucumber after inoculation with Pseudomonas syringae pv. syringae. Plant Physiol 1991, 97:1342-1347. 17. Durner J, Klessig DF: Nitric oxide as a signal in plants. Curr Opin Plant Biol 1999, 2:369-374. 18. Durner J, Wendehenne D, Kessig DF: Defense gene induction in tobacco by nitric oxide, cyclic GMP and cyclic ADP-ribose. Proc Natl Acad Sci USA 1998, 95:10328-10333. 19. Delledonne M, Xia Y, Dixon RA, Lamb C: Nitric oxide signal functions in plant disease resistance. Nature 1998, 394:585-588. 20. Alvarez ME, Pennell RI, Meijer P-J, Ishikawa A, Dixon RA, Lamb C: Reactive oxygen intermediates mediate a systemic signal network in the establishment of plant immunity. Cell 1998, 92:773-784. 21. Fritig B, Heitz T, Legrand M: Antimicrobial proteins in induced plant defense. Curr Opin Immunol 1998, 10:16-22. 22. Broglie K, Chet I, Holliday M, Cressman R, Biddle P, Knowlton S, Mauvais CJ, Broglie R: Transgenic plants with enhanced resistance to the fungal pathogen Rhizoctonia solani. Science 1991, 254:1194-1197. 23. Zhu Q, Maher EA, Masoud S, Dixon RA, Lamb CJ: Enhanced protection against fungal attack by constitutive co-expression of chitinase and glucanase genes in transgenic tobacco. Biotechnology 1994, 12:807-812. 24. Brunner F, Stintzi A, Fritig B, Legrand M: Substrate specificities of tobacco chitinases. Plant J 1998, 14:225-234.

Innate immunity in plants Cohn, Sessa and Martin 61 25. Bishop JG, Dean AM, Mitchell-Olds T: Rapid evolution in plant chitinases: molecular targets of selection in plant-pathogen coevolution. Proc Natl Acad Sci USA 2000, 97:5322-5327. Several studies have examined the possibility that resistance (R) genes have co-evolved with microbial pathogens, suggesting that pathogen perception is one of the most critical parameters that a plant must change in order to survive against much faster-evolving pathogens. This study presents a very interesting finding that a family of proteins that are induced subsequent to pathogen recognition are able to evolve quite rapidly. 26. Flor H: Current status of the gene-for-gene concept. Annu Rev Phytopathol 1971, 9:275-296. 27. Bendahamane A, Kanyuka K, Baulcombe DC: The Rx gene from potato controls separate virus resistance and cell death responses. Plant Cell 1999, 11:781-791. 28. Ellis JG, Lawrence GJ, Luck JE, Dodds PN: Identification of regions in alleles of the flax rust resistance gene L that determine differences in gene-for-gene specificity. Plant Cell 1999, 11:495-506. This report supports the theory that leucine-rich repeat (LRR) regions of some R gene products are directly involved in determining the specificity of pathogen perception. Sequence analysis of 13 alleles of the flax rust R gene, L, showed that specificity for different strains of flax rust is determined by differences in the coding region of the TIR (Toll/IL-1-receptor) and LRR domains of the various alleles. It is suggested that these alleles might have co-evolved with avirulence (Avr) genes of flax rust. 29. Warren RF, Henk A, Mowery P, Holub E, Innes RW: A mutation within the leucine-rich repeat domain of the Arabidopsis disease resistance gene RPS5 partially suppresses multiple bacterial and downy mildew resistance genes. Plant Cell 1998, 10:1439-1452. 30. Luck JE, Lawrence GJ, Dodds PN, Shepherd KW, Ellis JG: Regions outside of the leucine-rich repeats of flax rust resistance proteins play a role in specificity determination. Plant Cell 2000, 12:1367-1377. 31. Whitham S, Dinesh-Kumar SP, Choi D, Hehl R, Corr C, Baker B: The product of the tobacco mosaic virus resistance gene N: similarity to Toll and the interleukin-1 receptor. Cell 1994, 78:1011-1015. 32. Dinesh-Kumar SP, Baker BJ: Alternatively spliced N resistance gene transcripts: their possible role in tobacco mosaic virus resistance. Proc Natl Acad Sci USA 2000, 97:1908-1913. 33. Martin GB, Brommonschenkel S, Chunwongse J, Frary A, Ganal MW, Spivery R, Wu T, Earle ED, Tanksley SD: Map-based cloning of a protein kinase gene conferring disease resistance in tomato. Science 1993, 262:1432-1436. 34. Martin GB, Frary A, Wu T, Brommonschenkel S, Chunwongse J, Earle ED, Tanksleyk SD: A member of the tomato Pto gene family confers sensitivity to fenthion resulting in rapid cell death. Plant Cell 1994, 6:1543-1552. 35. Loh Y-T, Zhou J, Martin GB: The myristylation motif of Pto is not required for disease resistance. Mol Plant Microbe Interact 1998, 11:572-576. 36. Salmeron JM, Oldroyd GED, Rommens CMT, Scofield SR, Kim H-S, Lavelle DT, Dahlbeck D, Staskawicz BJ: Tomato Prf is a member of the leucine-rich repeat class of plant disease resistance genes and lies embedded within the Pto kinase gene cluster. Cell 1996, 86:123-133. 37. Joosten MHAJ, Cozijnsen TJ, de Wit PJGM: Host resistance to a fungal tomato pathogen lost by a single base-pair change in an avirulence gene. Nature 1994, 367:384-386. 38. Van Den Ackerveken GFJM, Van Kan JAL, de Wit PJGM: Molecular analysis of the avirulence gene avr9 of the fungal tomato pathogen Cladosporium fulvum fully supports the gene-for-gene hypothesis. Plant J 1992, 2:359-366. 39. Dixon MS, Jones DA, Keddie JS, Thomas CM, Harrison K, Jones JDG: The tomato Cf-2 dieases resistance locus comprises two funtional genes encoding leucine-rich repeat proteins. Cell 1996, 84:451-459. 40. Song W-Y, Wang G-L, Chen L-L, Kim H-S, Pi L-Y, Holsten T, Gardner J, Wang B, Zhai W-X, Zhu L-H et al.: A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21. Science 1995, 270:1804-1806. 41. Lease KIE, Walker JC: Challenges in understanding RLK function. Curr Opin Plant Biol 1998, 1:388-392. 42. He Z, Wang Z-Y, Li J, Zhu Q, Lamb C, Ronald R, Chory J: Perception of brassinosteroids by the extracellular domain of the receptor kinase BRl1. Science 2000, 288:2360-2363. An elegant study that used chimeric protein fusions to show that the extracellular domain of the receptor-like kinase (RLK) protein, BRl1, is involved in the perception of brassinosteroids. This work indicates that the kinase domain of leucine-rich repeat (LRR)-containing RLKs contributes to the specificity of the signalling pathway initiated and that the extracellular domain (possibly the LRR region) is directly involved in signal perception. The assay developed in this work should prove to be very useful in the identification of unknown ligands for members of the growing family of plant RLK proteins. 43. Galan JE, Collmer A: Type III secretion machines: bacterial devices for protein delivery into host cells. Science 1999, 284:1322-1328. A good review of type III secretion mechanisms used by pathogens of animals and plants. It is interesting to note that animal and plant pathogens share conserved mechanisms of delivery of effector molecules into host cells. In light of this, it is not so surprising that plants and animals may also share similar mechanisms to defend themselves against pathogens. 44. Scofield SR, Tobias CM, Rathjen JP, Chang JH, Lavelle DT, Michelmore RW, Staskawicz BJ: Molecular basis of gene-for-gene specificity in bacterial speck disease of tomato. Science 1996, 274:2063-2065. 45. Tang X, Frederick RD, Zhou J, Halterman DA, Jia Y, Martin GB: Initiation of plant disease resistance by physical interaction of AvirulencePto and Pto kinase. Science 1996, 274:2060-2063. 46. Jia Y, McAdams SA, Bryan GT, Hershey HP, Valent B: Direct interaction of resistance gene and avirulence gene products confers rice blast resistance. EMBO J 2000, 19:4004-4014. This study is the first report of direct interaction between a fungal Avr protein and the product of an R gene. The authors clearly demonstrate this interaction using the yeast two-hybrid system, as well as demonstrating interaction in vitro. The authors suggest that Avr-Pita may be introduced by the fungus into the plant cell, where it interacts with the predicted cytoplasmic Pita protein via an as-yet uncharacterized secretion mechanism. 47. Leister RT, Katagiri F: A resistance gene product of the nucleotide binding site-leucine rich repeats class can form a complex with bacterial avirulence proteins in vivo. Plant J 2000, 22:345-354. Despite the number of reports that have provided circumstantial evidence that Avr proteins interact with R proteins in vivo, no studies previous to this one have provided clear biochemical evidence of this interaction. It should be noted, however, that the assay used in this study also detected the interaction of RPS2 and AvrB, two proteins that should not interact directly if the ligand receptor model of gene-for-gene specificity holds true. 48. Zhu W, Yang B, Kurata N, Johnson LB, White FF: The C terminus of AvirulenceXa10 can be replaced by the transcriptional activation domain of VP16 from the herpes simplex virus. Plant Cell 1999, 11:1665-1674. 49. Nimchuk Z, Marois E, Kjemtrup S, Leister RT, Katagiri F, Dangl JL: Eukaryotic fatty acylation drives plasma membrane targeting and enhances function of several type III effector proteins from Pseudomonas syringae. Cell 2000, 101:353-363. Several avirulence (Avr) proteins have been shown to possess consensus myristylation sites; however, the role of these sites was unclear. This study shows that the myristylation motif of the Avr proteins is required for efficient localisation to the host plasma membrane. The bacterial Avr proteins were actually acylated in planta and this host-dependent post-translational modification was required for proper localisation and maximal activity of the protein effector molecules. 50. Shan L, Thara VK, Martin GB, Zhou J-M, Tang X: Pseudomonas AvrPto protein is differentially recognized by tomato and tobacco and is localized to the plant plasma membrane. Plant Cell 2000, 12:in press. This report provides evidence that an avirulence (Avr) protein is directed to the plasma membrane via myristylation inside the plant cell. Mutation of a putative myristylation motif of AvrPto was shown to abolish its avirulence activity. Additionally, the authors showed, using point-mutational analysis, that the AvrPto protein is differentially recognized in tomato and tobacco. 51. Hatada EN, Krappmann D, Scheidereit C: NF-κB and the innate immune response. Curr Opin Immunol 2000, 12:52-58. Activation of members of the family of Rel/NF-κB transcription factors is a central theme of innate immunity in mammals and insects. This review covers many of the recent findings of studies of the IL-1-receptor/Toll signalling pathways. A better understanding of how transactivation of defence-related proteins is activated in this pathway is likely to provide clues about the activation of defence genes in plants.

62 Innate immunity 52. Sessa G, D Ascenzo M, Loh y-t, Martin GB: Biochemical properties of two protein kinases involved in disease resistance signaling in tomato. J Biol Chem 1998, 273:15860-15865. 53. Sessa G, D Ascenzo M, Martin GB: Thr38 and Ser198 are Pto autophosphorylation sites required for the AvirulencePto-Pto mediated hypersensitive response. EMBO J 2000, 19:2257-2269. A detailed biochemical analysis of the autophosphorylation activity of the kinase, Pto. Specific residues were identified that were critical for interaction with the ligand AvrPto and with downstream signalling molecules. This report indicates that autophosphorylation is needed for biological activity and that phosphorylation of specific residues might affect interaction with proteins involved in distinct signaltransduction mechanisms. Remarkably, Thr38, which is the main autophosphorylation site required for Pto function, is conserved in both the human IRAK (IL-1-receptor-associated kinase) and the Drosophila Pelle proteins. 54. Frederick RD, Thilmony RL, Sessa G, Martin GB: Recognition specificity for the bacterial avirulence protein AvrPto is determined by Thr204 in the activation loop of the tomato Pto kinase. Mol Cell 1998, 2:241-245. 55. Rathjen JP, Chang JH, Staskawicz BJ, Michelmore RW: Constitutively active Pto induces a Prf-dependent hypersensitive response in the absence of AvrPto. EMBO J 1999, 18:3232-3240. 56. Zhou J, Loh Y-T, Bressan RA, Martin GB: The tomato gene Pti1 encodes a serine/threonine kinase that is phosphorylated by Pto and is involved in the plant hypersensitive response. Cell 1995, 83:925-935. 57. Ligterink W, Kroj T, zur Nieden U, Hirt H, Scheel D: Receptormediated activation of a MAP kinase in pathogen defense of plants. Science 1997, 276:2054-2057. 58. Zhang S, Klessig DF: N resistance gene-mediated de novo synthesis and activation of a tobacco MAP kinase by TMV infection. Proc Natl Acad Sci USA 1998, 95:7433-7438. 59. Romeis T, Piedras P, Jones JDG: Resistance gene-dependent activation of a calcium-dependent protein kinase in the plant defense response. Plant Cell 2000, 12:803-815. This is the first report that a CDPK is involved in resistance-gene-mediated plant defence signalling. Several CDPKs have been isolated from plants and they appear to act as signal molecules involved in a variety of signal-transduction pathways in which Ca 2+ is a second messenger. CDPKs may be analogous to PKC isomers described in animal signalling pathways. 60. Zhou J, Tang X, Martin GB: The Pto kinase confering resistance to tomato bacterial speck disease interacts with proteins that bind a cis-element of pathogenesis-related genes. EMBO J 1997, 16:3207-3218. 61. Gu Y-QY, Yang C, Thara VK, Zhou J, Martin GB: The Pti4 gene is regulated by ethylene and salicylic acid and its product is phophorylated by the Pto kinase. Plant Cell 2000, 12:771-785. Little is presently known about how R-gene-mediated signalling pathways activate the transcription of pathogenesis resisitance (PR) genes. This report describes a link between the activation of a resistance protein and the regulation of a transcription factor that binds to promoter regions of PR genes. 62. Thara VK, Tang X, Gu YQ, Martin GB, Zhou J-M: Pseudomonas syringae pv. tomato induces the expression of tomato EREBP-like genes Pti4 and Pti5 independent of ethylene, salicylate, and jasmonate. Plant J 1999, 20:475-483. 63. Cao H, Glazebrook J, Clarke JD, Volko S, Dong X: The Arabidopsis NPR1 gene that controls systemic aquired resistance encodes a novel protein containing ankyrin repeats. Cell 1997, 88:57-63. 64. Ryals JA, Weymann K, Lawton K, Friedrich L, Ellis D, Steiner HY, Johnson J, Delaney TP, Jesse T, Vox P et al.: The Arabidopsis NIM1 protein shows homology to the mammalian transcription factor inhibitor IkB. Plant Cell 1997, 9:425-439. 65. Zhang Y, Fan W, Kinkema M, Li X, Dong X: Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene. Proc Natl Acad Sci USA 1999, 96:6523-6528. One of three recent reports (with [66,67 ]) that used the yeast two-hybrid system to search for proteins that interact with the NPR1 protein. It was found that NPR1 interacts with transcription factors that are believed to be involved in the activation of pathogenesis-related (PR) genes. These findings represent very significant findings in the understanding of how pathogen recognition by resistance (R) genes may control defence gene transcription. 66. Despres C, DeLong C, Glaze S, Liu E, Fobert PR: The Arabidopsis NPR1/NIM1 protein enhances the DNA binding activity of a subgroup of the TGA family of bzip transcription factors. Plant Cell 2000, 12:279-290. See annotation to [65 ]. 67. Zhou J-M, Trifa Y, Silva H, Pontier D, Lam E, Shah J, Klessig DF: NPR1 differentially interacts with members of the TGA/OBF family of transcription factors that bind an element of the PR-1 gene required for induction by salicylic acid. Mol Plant Microbe Interact 2000, 13:191-202. See annotation to [65 ].