Targeting of protein signals from Xanthomonas to the plant nucleus

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the enzyme ACC oxidase (the activity of which leads to the formation of ethylene) accumulate in cell layers opposite the phloem poles (Fig. 2). Because nodule primordia are normally found predominantly opposite the protoxylem poles, these results suggest that the cell divisions leading to the formation of nodule primordia are negatively regulated by a gradient of ethylene governed by the activity of ACC oxidase. Consistent with this hypothesis, it was shown that the external addition of AVG or silver leads to an increased number of nodule primordia located opposite the phloem poles. It is likely that a negative feedback mechanism operating via ethylene is regulated by Nod factors, because these have been shown to be capable of inducing various ethylene-related effects in pea roots 9. It was also shown that ethylene is not necessary for the positive effect of nodulation factors on polar tip growth of root hairs in vetch (Vicia spp.) 5. These effects on polar tip growth are probably related to processes involved in the initial steps of the infection process in vetch; these include the formation of curled root hairs and preinfection threads in the outer cortex4. In contrast, ethylene appears to be required for the formation of root hairs and their subsequent outgrowth in vetch, consistent with the proposed role of ethylene during root hair development in Arabidopsis 13.

Linking the findings It will be interesting to link these findings with information on the processes of root hair curling and pre-infection thread formation in the skl mutant. However, further analysis of the action of ethylene in the

infection and nodulation process will remain difficult in the absence of detailed knowledge of the components of the signal transduction pathway. This objective could benefit greatly from the rapid advances in studies on ethylene signal transduction in the model plant, Arabidopsis 11-14. The importance of sustained research into the role of ethylene in leguminous plant systems lies in the relevance of this simple gas as an antagonist of complex developmental processes, and this could be relevant to many other types of plant-microbe interactions2.

Herman P. Spaink Institute of Molecular Plant Sciences, Leiden University, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands (tel +31 71 527 5055; fax +31 71 527 5085; e-mail spaink@ rulsfb.leidenuniv.nl)

References 1 Spaink, H.P. (1996) Regulation of plant morphogenesisby lipo-chitin oligosaccharides, Crit. Rev. Plant Sci. 15, 559-582 2 Spaink, H.P. (1995) The molecular basis of infection and nodulation by rhizobia: the ins and outs of sympathogenesis, Annu. Rev. Phytopathol. 33, 345-368 3 Caetano-Anoll~s,G. and Gresshoff, P.M. (1991) Plant genetic control of nodulation, Annu. Rev. Microbiol. 45, 345-382 4 van Brussel, A.A.N.et al. (1992) Induction of pre-infection thread structures in the leguminous host plant by mitogenic lipooligosaccharides of Rhizobium, Science 257, 70-72 5 Heidstra, R. et al. Ethylene provides positional information on cortical cell division but is not involved in Nod factor induced tip growth, Development (in press)

6 Penmetsa, R.V. and Cook, D.R. (1997) A legume ethylene-insensitive mutant hyperinfected by its rhizobial symbiont, Science 275, 527-530 7 Fearn, J.C. and Larue, T.A. (1991) Ethylene inhibitors restore nodulation to sym5 mutants ofPisum sativum L. cv. Sparkle, Plant Physiol. 96, 239-244 8 Lee, K.H. and Larue, T.A. (1992) Exogenous ethylene inhibits nodulation of Pisum sativum L. cv. Sparkle, Plant Physiol. 100, 1759-1763 9 van Spronsen, P.C., van Brussel, A.A.N.and Kijue, J.W. (1995) Nod factors produced by Rhizobium leguminosarum biovar, vieiae induce ethylene-related changes in root cortical cells of Vicia sativa ssp. nigra, Eur. J. Cell Biol. 68, 463-469 16 Zaat, S.A.J. et al. (1989) The ethyleneinhibitor aminoethoxyvinylglycine restores normal nodulation by Rhizobium leguminosarum biovar, viciae on Vicia sativa subsp, nigra by suppressing the 'Thick and short roots' phenotype, Planta 177, 141-150 11 Hua, J. et al. (1995) Ethylene insensitivity conferred by Arabidopsis ERS gene, Science 269, 1712-1714 12 Roman, G. et al. (1995) Genetic analysis of ethylene signal transduction in Arabidopsis thaliana: five novel mutant loci integrated into a stress response pathway, Genetics 139, 1393-1409 13 Tanimoto, M., Roberts, K. and Dolan, L. (1995) Ethylene is a positive regulator of root hair development in Arabidopsis thaliana, Plant J. 8, 943-948 14 Bleecker, A.B. (1991) Genetic analysis of ethylene responses in Arabidopsis thaliana, Syrup. Soc. Exp. Biol. 45, 149-158

Targeting of protein signals from Xanthomonas to the plant nucleus Until last year, the only microbial plant pathogens that were known to transfer large molecules into the plant cell, let alone the nucleus, were in the bacterial family Rhizobiaceae. Pathogenicity of Agrobacterium tumefaciens is determined by its capacity to transfer a piece of its DNA, the T-DNA, into plant nuclei. The methods by which plant pathogenic bacteria outside the Rhizobiaceae were thought to condition disease were generally assumed to be less complicated. The various rots, blights, leaf spots, wilts, cankers and the like were thought to be induced by enzymes and signals that were secreted to the outside of the plant cell wall. Then came the surprising discovery of functional nuclear localization signal sequences encoded by a large family of Xanthomonas avr/pth ('avirulence and 204

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pathogenicity') genes, which are required for such diverse and host-specific plant responses as cell division, water soaking (i.e. filling of the intercellular spaces in the leaf mesophyll with water instead of air) and the hypersensitive response 1. This first indication that bacterial pathogens other than Agrobacterium might deliver proteins directly into the plant cell was followed by evidence of signal transfer from four independent research groups uS. The proteins encoded by avr genes from both Xanthomonas and Pseudomonas signal the hypersensitive response directly when expressed inside the plant cell. One such signal protein, AvrPto (Pto, 'Pseudomonas syringae pv. tomato'), was shown to bind to a cytoplasmic receptor protein kinase encoded by the

plant resistance gene Pto (Refs 4 and 5), demonstrating that there is a protein ligand-receptor mechanism for signal transduction during the hypersensitive response. In a recent paper in Cell, it was revealed that the AvrBs3 (Bs, 'bacterial speck') protein functions inside the plant cell2. This realization is especially significant, because avrBs3 is a member of the Xanthomonas avr/pth gene family, and most known members of this family function as pth genes. Mutations in the nuclear localization signal sequences of avrBs3 (Ref. 2) and two other members of the avr/pth gene family, pthA (A, 'Asiatic Citrus canker') (Ref. 6) and avrb6 (Ref. 6) reduced nuclear localization, avirulence encoded by avrBs3 and avrb6, and pathogenicity on Citrus encoded by pthA. Taken

© 1997 Elsevier Science Ltd

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together, these data indicate that not only avirulence, but also the pathogenicity of several xanthomonads, is determined in part by protein signals that are delivered and perceived inside the plant cell.

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Functional nuclear localization signals encoded by Xanthomonas pathogenicity genes Necrotrophic gram-negative bacteria, which derive nutrition from dead or dying plants, rely heavily on the secretion of degradative enzymes and toxins to kill cells, and do not establish extensive contact with living cells. Even the biotrophic gramnegative bacterial pathogens (in the genera Erwinia, Pseudomonas and Xanthomonas), which establish extensive host-cell contact, were previously thought to rely on toolecules secreted outside the plant cell wall to establish a parasitic relationship. Both necrotrophs and biotrophs use a novel type III protein secretion system found only in pathogens, and encoded by about 16 hrp ('hypersensitive response and pathogenicity') genes 7's. In contrast with type I and type II systems, type III secretion is triggered when a pathogen comes into close contact with host cells ~. Not surprisingly, the hrp genes play an essential role in the pathogenicity of all biotrophic Erwinia, Pseudomonas and Xanthomonas strains. The hrp genes were so named because mutations in them abolish all pathogenic phenotypes - not only the ability to grow and elicit pathogenic symptoms on all host species, but also the ability to elicit a plant hypersensitive response, indicative or: cell deathT,1°. Only a few proteins were known to be secreted by the hrp genes, but prominent among them is a group of proteinaceous elicitors generically referred to as 'harpins'. Harpins are essential for the pathogenicity of Erwinia and Pseudomonas and, when applied to plant cells extracellularly, elicit many of the same responses as the pathogen from which they were extracted (although they are not particularly host species-specificT). Despite intensive efforts in several labs, no harpins or harpin-like pathogenicity factors have been found in the genus Xanthomonas, raising the question as to how pathogenicity is achieved by Xanthomonas and what effector molecules are secreted by its hrp system. Xanthomonas differs from Pseudomonas and Erwinia in that: • All xanthomonads are associated with plants. • All xanthomonads are biotrophic. • Each individual Xanthomonas strain typically exhibits a much higher level of plant species specificity than strains of the other two genera. The few non-hrp pathogenicity genes described from Xanthomonas, such as pthA (which is required for Citrus canker disease) and avrb6 (which contributes to cotton blight disease) are host-species

34-amino-acid leucine-rich repeats .................... encoded by the gene determine the plant response phenotype

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Citrus-specific hyperplasia

Cotton-specific water soaking

Rice-specific lesions

Cultivar-specific hypersensitive response (hosts and nonhosts)

Fig. 1. The general structure of the nearly identical genes in the Xanthomonas avr/pth ('avirulence and pathogenicity') gene family. When expressed in Xanthomonas containing a functional type III secretion system encoded by hrp ('hypersensitive response and pathogenicity') gene(s), the plant response phenotype depends on the exact sequence of the 34-amino-acid leucine-rich repeats encoded by the avr/pth gene. The three currently known plant species-specific pathogenicity phenotypes, as well as the cultivar-specific avirulence phenotype, are shown (although there are probably more species-specific pathogenicity phenotypes). Hyperplasia is pathogen-induced cell division; with Citrus canker disease, mesophyll cells are caused by the pathogen to divide in sufficient numbers to rupture the epidermal cell layer, resulting in canker lesions. Water soaking is a pathogeninduced filling of the intercellular spaces in the leaf mesophyll with water instead of air. Based on the recent demonstration that avrBs3 expression inside the plant cell is sufficient to elicit the cultivar-specific hypersensitive response 2, and other data involving site-directed mutations of the nuclear localization signals, the indicated pathogenicity phenotypes also appear to be determined inside the plant cell by the proteins encoded by other members of the avr/pth gene family.

specific in function. Although the phenotypes determined by these genes are hrpdependent, their protein products were certainly not considered as primary candidates for secretion. The discovery of functional nuclear localization signal sequences in the predicted proteins encoded bypthA and avrb6 (Ref. 1) was quite unexpected. Could the host species-specific pth genes of Xanthomonas, which are capable of eliciting such diverse phenotypes as cell division in Citrus and water soaking in cotton, encode signaling proteins that are secreted from Xanthomonas and condition pathogenicity by affecting plant cell programs from inside the plant cell?

The Xanthomonas avr/pth gene family and avirulence Both pthA and avrb6 are members of a large Xanthomonas avr/pth gene family; all members sequenced to date share >90% identity at the nucleotide level. The most conspicuous feature of the gene family is the presence, in the central region of the predicted proteins, of a dozen or more nearly identical, tandemly arranged, leucine-rich, 34-amino-acid direct repeats (Fig. 1). These repeats determine both the cultivar-specific avirulence and speciesspecific pathogenicity functions 11. Most members of this gene family were originally isolated as avr genes (defined as genes

causing avirulence on host cultivars carrying resistance genes), but avirulence can hardly be a purposeful function in a plant pathogen. In fact, a pathogenicity function has been established for a majority of the known members, including avrb6, avrb7, avrBIn, avrBl01, avrBl02, avrBl04 and avrB5 (water soaking of cotton) 12,pthA (cell division of Citrus)13, and avrXa7 (Xa, 'Xanthomonas') (elongated lesions of rice) 14. Members of the gene family are widely distributed in the genus, but not all pathogenic xanthomonads carry members, demontrating that the genes are not universally required for Xanthomonas pathogenicity. Even when they are present, some members, such as avrBs3, appear to confer only cultivar-specific avirulence and are thus entirely dispensable, lacking any detected pleiotropic function. Many, such as avrBs3, are not conserved as alleles within their pathogenic group (in this case, X. campestris pv. vesicatoria). Such avr genes therefore appear to lack a pathogenic function, and are not only dispensable, but their presence appears to be gratuitous. Virulence on plants carrying resistance genes is achieved by the mutation or loss of such avr genes. Direct evidence for secretion of Xanthomonas Avr/Pth proteins is still required. Studies in two independent tabs have shown, by immunocytochemistry, June1997,Vol.2, No,6

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that the protein products of two members of the gene family are located within the cytoplasm of the pathogen following inoculation into plants 15. No evidence was found for secretion in either study. Furthermore, another member of the gene family, avrBs3-2 from X. campestris pv. vesicatoria, still functioned for avirulence after a large portion of the 3' end of the gene including the nuclear localization signalencoding region - was deleted. This demonstrated that AvrBs3-2 does not require a functional nuclear localization Signal to confer avirulence. However, detailed deletion analyses and transposon saturation mutagenesis of at least 10 other members of the a v r / p t h gene family, including avrBs3, avrb6 and pthA, demonstrated that the regions of these genes that encode the nuclear localization signals are functional requirements for avirulence 1.

Nuclear localization signals and pathogenic/avirulence phenotypes Site-directed mutations of the nuclear localization signal sequences in pthA reduced the nuclear localization function (in onion cells), and also reduced the pathogenicity (hyperplastic cankers on Citrus) conferred by the gene G. Similar experiments using avrBs3 have confirmed the nuclear localization function in onion cells, and role in the avirulence (assayed as a visible hypersensitive response on hosts) phenotype conferred by this gene 2. This provided strong, but indirect, evidence that the PthA and AvrBs3 proteins were translocated into host plant cells and were targeted to the cell nucleus. Evidence that AvrBs3 is both necessary and sufficient to elicit a hypersensitive response once inside host cells was obtained when the avrBs3 coding region was recloned, under the control of a plant promoter and using the TDNA ofA. tumefaciens, and inoculated onto plants 2. A hypersensitive response was observed that could only be explained by expression of avrBs3 from the T-DNA inside the plant cell. The extent of the role played by the hrp genes in translocation through the plant cell wall is unclear. In one model, the hrp type III secretion system delivers the Avr/Pth protein signals to the cell surface and receptor-mediated endocytosis then transports the proteins into the plant cell1. In another model, the Avr/Pth proteins are proposed to be transported through a hollow, pilus-like structure produced by the products of the hrp genes 2. However, the proteins pass across the plasma membrane and, although the evidence is indirect, it seems likely that once inside they are translocated to the nucleus. The lack of direct evidence for secretion of these Avr/Pth protein signals may be for two reasons. First, type III secretion is different to types I and II, in that type III secretion is contact-dependent and may only occur at regions of the bacterial cell

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wall that are in direct contact with the plant cell wall. Secretion of signal factors into the external environment would be wasteful. Therefore, it is possible that these proteins are secreted only to, or through, the plant cell wall. Second, it is possible that the immunolocalization experiments described failed to detect the signal proteins because the proteins were secreted in amounts below the threshold limits of detection. In both studies, the genes were deliberately overexpressed to allow detectionTM. Direct evidence for the involvement of nuclear localization signal sequences in proteins involved in T-DNA transfer by Agrobacterium is also lacking.

New prospects for bacterial disease control The finding that a major pathogenicity gene family in Xanthomonas encodes pro-

tein signal molecules that enter a plant cell suggests that there may be entirely novel means of controlling some of the diseases caused by Xanthomonas and other biotrophic pathogens. For example, there is strong evidence that Citrus canker, bacterial rice blight and bacterial cotton blight all require members of the a v r / p t h gene family for pathogenicity. Several of the most effective genetic methods for controlling plant virus diseases came not as a result of cloning the available virus resistance genes, but instead as a result of understanding the function of virus genes and then using that knowledge to block essential virus functions. No doubt, novel disease control strategies, not necessarily involving the cloning of available plant resistance genes, but instead based on an understanding of the signal molecules that condition pathogenicity, are now under development. A search for the effector molecules secreted by the hrp genes in Erwinia, Pseudomonas and Xanthomonas would appear to be warranted.

Acknowledgements The author would like to record this article as number R-05714 from the Florida Agricultural Experimental Station.

Dean W. Gabriel Plant Molecular and Cell Biology Program, Plant Pathology Dept, University of Florida, Gainesville, FL 32611-0680, USA (tel +1 352 392 7239; fax +1 352 392 6532;

e-mail [email protected]) References 1 Yang, Y. and Gabriel, D.W. (1995) Xanthomonas avirulence/pathogenicity gene family encodes functional plant nuclear targeting signals, Mol. Plant-Microbe Interact. 8, 627-631 2 Van den Ackerveken, G., Marois, E. and Bonas, U. (1996) Recognition of the bacterial avirulence protein AvrBs3 occurs inside the host plant cell, Cell 87, 1307-1316

3 Gopalan, S. et al. (1996) Expression of the Pseudomonas syringae avirulence protein AvrB in plant cells alleviates its dependence on the hypersensitive response and pathogenicity (Hrp) secretion system in eliciting genotype-specifichypersensitive cell death, Plant Cell 8, 1095-1105 4 Tang, X. et al. (1996) Initiation of plant disease resistance by physical interaction of AvrPto and Pto kinase, Science 274, 2060-2063 5 Scofield,S.R. et al. (1996) Molecular basis of gene-for-genespecificity in bacterial speck disease of tomato, Science 274, 2063-2065 6 Gabriel, D.W. et al. (1996) Role of nuclear localizing signal sequences in three disease phenotypes determined by the Xanthomonas avr/pth gene family, in Biology of Plant-Microbe Interactions (Stacey, G., Mullin, B. and Gresshoff, P.M., eds), pp. 197-202, International Society for Molecular Plant-Microbe Interactions 7 Alfano, J.R. and Collmer, A. (1997) Bacterial pathogens in plants: life up against the wall, Plant Cell 8, 1683-1698 8 Van Gijsegem, F., Genin, S. and Boucher, C. (1993) Conservation of secretion pathways for pathogenicity determinants of plant and animal bacteria, Trends Microbiol. 1, 175-180 9 Mecsas, J. and Strauss, E.J. (1996) Molecular mechanisms of bacterial virulence: type III secretion and pathogenicity islands, Emerg. Infect. Dis. 2, 271-288 10 Dangl, J.L., Dietrich, R.A. and Richberg, M.H. (1996) Death don't have no mercy: cell death programs in plant-microbe interactions, Plant Cell 8, 793-1807 11 Yang, Y., De Feyter, R. and Gabriel, D.W. (1994) Host-specificsymptoms and increased release ofXanthomonas citri and X. campestris pv. malvacearum from leaves are determined by the 102 bp tandem repeats of pthA and avrb6, respectively, Mol. Plant-Microbe Interact. 7, 345-355 12 Yang, Y., Yuan, Q. and Gabriel, D.W. (1996) Water soaking function(s) of XcmH1005 are redundantly encoded by members of the Xanthornonas avr /pth gene family, Mol. Plant-Microbe Interact. 9, 105-113 13 Swarnp, S. et al. (1992) A Xanthomonas citri pathogenicity gene, pthA, pleiotropically encodes gratuitous avirulence on nonhosts, Mol. Plant-Microbe Interact. 5, 204-213 14 Leach, J.E. et al. (1996) Genes and proteins involved in aggressiveness and avirulence of Xanthornonas oryzae pv. oryzae to rice, in Biology of Plant-Microbe Interactions (Stacey, G., Mullin, B. and Gresshoff, P.M., eds), pp. 191-196, International Society for Molecular Plant-Microbe Interactions 15 Bonas, U., Conrads-Strauch, J. and Balbo, I. (1993) Resistance in tomato to Xanthomonas campestris pv. vesicatoria is determined by alleles of the pepper-specific avirulence gene avrBs3, Mol. Gen. Genet. 238, 261-269 16 Leach, J.E. and White, F.F. (1996) Bacterial avirulence genes, Annu. Rev. Phytopathol. 34, 153-179