- Email: [email protected]
Plant-cyst nematode and plant-root-knot nematode interactions
Parasitology Today, vol. IO, no. I I, I994
424
Plant-Cyst Nematode and Plant-Root-knot Nematode Interactions A. Niebel, G. Gheysen and M. Van Montagu Root-knot nematodes and cyst nematodes are obligate plant parasites that cause extensive damage to the agriculture of both temperate and tropical countries. In this review, Andreas Niebel, Godelieve Gheysen and Marc Van Montagu describe how, in the past decade, the use of molecular techniques has provided new insights in the complex interactions between these sedentary plant-parasitic nematodes and their infected host plants. They give an account of the progress in OUY understanding of both the parasite and the host during compatible and incompatible interactions. They also outline the importance of a new model host system, Arabidopsis thaliana. Among plant-parasitic nematodes, the root-knot nematodes (Meloidogyninae) and the cyst nematodes (Heteroderinae) have, over the past decade, received increasing attention. This is largely a consequence of the agronomic impact of one root-knot nematode genus (Meloidogyne) and two cyst nematode genera (Heterodera and Globoderu). Meloidogyne is the most destructive plant-parasitic nematode genus in the world. It infects over 2000 plant species, contributing greatly to an estimated annual yield loss of US$77 billionl. Although Meloidogyne is nearly ubiquitous, it represents a particular threat to agriculture in tropical and sub-tropical regions. In contrast, Heteroderu and Globoderu are the greatest problem for agriculture in more temperate regions. Both types of plant-parasitic nematodes are obligate parasites, spending the major part of their life cycle within roots (Figs 1 and 2). In common with some animal parasites, such as Trichinella spirulis2, they do not kill the host cells from which they feed. Instead, the nematodes induce a redifferentiation process that leads to the formation of multinucleated feeding cells on which they depend for the completion of their life cycle (Figs l-3). These feeding cells appear to be metabolically active: their cytoplasm is very dense, containing numerous mitochondria, plastids, ribosomes, welldeveloped Golgi apparatus, and smooth endoplasmic reticulum, generally organized in swirls. The central vacuole disappears and gives rise to many small vacuoles; nuclei and nucleoli become hypertrophic and assume an amoeboid profilea-5. In addition, special secondary cell wall formations develop (Fig. 4). These are called ‘cell wall ingrowths’ and are typical of transfer cell+. Cell wall ingrowths are thought to enhance solute uptake from the vascular system7. Parallels can be drawn with T. spiralis, where a plexus of venules, called placenta, surrounds the parasitized muscle cells, probably to facilitate transport between the parasite and its hostz. Although feeding cells Andreas Niebel, Godelieve Gheysen and Marc Van Montagu are at the Labotatotium voor Genetica, Univeniteit Gent B-9000 Gent, Belgium.
induced by root-knot and cyst nematodes are similar in ultrastructure and possibly in function, they are thought to be formed by distinct mechanisms. Cyst nematodes have been shown to induce a single, multinucleated syncytium by cell wall breakdown. Despite a long controversy gs, it is now generally accepted that cell wall breakdown plays no role in the formation of root-knot nematode-induced feeding cells. In this case, the parasite induces several ‘giant cells’ by repeated mitosis without CytokinesisiQli. Studies of compatible plant-nematode interactions Although the descriptive knowledge of plant-nematode interactions is extensive, little is known about the underlying biochemical and molecular events leading to the observed redifferentiation processes. Some years ago, several molecular approaches were initiated to identify and characterize plant genes altered in expression after infection by root-knot or cyst nematodes. The main problem in analyzing gene expression at nematode feeding sites is the limited number of root cells affected by the nematode. To circumvent this problem, Gurr et al.12 developed a polymerase chain reaction @‘CR)-based method that allowed them to construct a cDNA library from small amounts of Globoderu-infected potato root sectors (at the adult stage of the parasite, 26 days after inoculation). They then differentially screened the library with probes derived from infected and uninfected roots. One differentially expressed clone, pl’MR1, was induced locally within four days of inoculation. It showed no sequence homology with known sequences. To understand the role of pPMR1 in the plant-nematode interaction, further work is required, specifically, detailed expression analysis and precise cellular localization of the transcript. In our laboratory, a cDNA library was constructed using G. pallidu-infected potato roots (at the adult stage of the parasite) and differentially screened. Several G. pallida-induced potato genes were identifiedis. Sequence identity indicates that one of these, Nem2, encodes a potato catalase. Expression analysis showed that Nem2 is gradually induced after Globoderu attack and reaches its strongest expression in roots and stems of infected plants, approximately four weeks after inoculation. Other nematodes (M. incognita), as well as bacterial root pathogens (Enuiniu curotovoru and Coynebacterium sepedonicum), also induce this catalase in potato roots and stems. Catalase is a peroxisomal heme protein catalyzing the dismutation of hydrogen peroxide into water and oxygen. Hydrogen peroxide functions as a defense-related signal transducer, triggering plant defense mechanism+. In addition, the inhibition of catalase by salicylate has been shown to lead to a hydrogen peroxide-mediated systemic acquired resistance phenomenonis. We believe that compatible root pathogens in general, 0
1994, Elsev~er Swnce
Ltd
Porasiro/ogy
Today, vol.
IO, no. I I, I994
425
Fig. 1. Life cycle of a cyst nematode. (I) The larvae of the second stage (a) are attracted to the roots. After penetration, they migrate intmcellulorfy, direct/y toward the vascular cylinder. There, they start feeding upon a sing/e cell, which is rapid/y turned into a large syncytium by incorporation of neighbouring tissue (through cell wall breakdown). (2) The larvae undergo three additional moults.Here o female (b) and a mole (c) of the fourth larvul stage are shown. Whereas the body of the larvae bulges out of the root, their head remains embedded witlllin the root for food withdrawal from the syncytium. (3) During the last mou/t, the ma/e (e) changes shape dmm&a//y, /eaves the root, fenti/izes the female (d), and then dies. (4) At moturity, the cuticle of the fimole hardens and turns into a cyst (f) containing the eggs. Inside the eggs, the]/ larvae undergo their first moulc The 12 larvae (a) then hatch, often under the influence of specific substances present in root exudates. Depending on environmental conditions,the cycleis completed in one to two months.
and nematodes in particular, might have evolved mechanisms to increase plant catalase levels, thereby inhibiting or retarding hydrogen peroxide-mediated defense reactions. The role of the structural cell wall protein, extensin, during nematode infecuon has been studied in detail in tobacco*6. Extensin, one of the most abundant structural cell wall proteins, is thought to play a role in the termination of cell expansion and, thus, in cell wall rigidity, by being crosslinked either to itself or to other cell wall components*7. Extensin is induced in actively dividing cells (where the synthesis of new cell walls requires new structural material)ls, following compatible and incompatible pathogen attack19 and also several abiotic stresses. The tobacco cyst nematode, G. tubacum, only weakly induces extensin expression, probably due to wounding during penetration and migration through the roots. High extensin expression is observed in galls induced by M. juvanicu, especially during the second larval stage. The highest extensin expression can be found in the gall cortex. These cells may need to increase the rigidity of their cell walls in order to resist the mechanical pressure created by dividing pericycle cells (that also show significant extensin expression) and growing giant cells and nematodes. No significant amounts of extensin could
be found in the large cell walls of giant cells and particularly not in the cell wall ingrowths. This may illustrate the need for a loose cell wall structure (achieved in the absence of a dense protein network) for efficient solute uptake from the vascular system. It also indicates the ‘extensible’ state of the cell walls of expanding giant cells. A member of a membrane protein family, pRB7, believed to function as water channels, has recently been shown to be up-regulated in giant cells induced by different species of Meloidogyne2’J. Using transgenic plants containing the fobRB7 (the gene encoding pRB7) promoter fused to the coding sequence of the j3-glucuronidase A (gus) reporter gene, the authors could show that tobRB7 is transcribed in and around giant cells from four days until 40 days after inoculation. The expression in control roots is restricted to root me&terns and immature vascular cylinder regions. Promoter deletion studies showed that the root-specific expression pattern could be uncoupled from the nematode-induced gene expression. A 299bp S-flanking region was shown to confer giant cellspecific expression. The pRB7 protein might be important in regulating the water status of giant cells once their complex secondary cell wall and membrane modifications have developed. Interestingly, tobRB7 is
Fig. 2. Life cycle of o root-knot nematode. (I) The larvae of the second stage (0) are attracted to the roots. They usually penetrote the roots closely behind the root tip. The larvae then migrate first towards the root tip, where the absence ofdiferentiated endodermis allows them to enter the vascular cylinder. This migration happens intercellular/y by mechanical and possibly enzymatic sofiening of the middle lame/la. The parasites finaIry start feeding on three to ten cells, which are rapidly turned into mu/tinucleated giant cells, by endomitosis and cell hypertrophy. (2) At the same time as the giant cells are firmed, the ce//.sof the neighbouring pericyc/e start to divide, giving rise to a typica/ go// or root-knot Inside the gal/, D female (b) and o mole (c) of the 13 larval stage ore shown. (3) The gall continues to swell, while females (d) and moles (e) ore in their j4 stage. (4) During the last mou/t, the ma/e (h) dmmoticolly changes its shape, then leaves the root and fertilizes the female (f) in the case ofomphimictic species, However, parthenogenesis is often encountered in root-knot nemutodes. The female lc~ysits eggs in a gelatinous matrix (g) outside the root From there, the larvae of the second stage (a) hatch and are ottmcted to roots. Depending on environmental conditions, this cyc/e is completed in one to two months.
Parasitology Today, vol. IO, no. I I, I 994
426
b Fig. 3. Light micrograph of a cross-section through a potato root infected by the potato cyst nematode Globodera pallida. (a) The head of the nematode (N) inserted into the root is clearly visible, whereas only the distal part of the induced syncytium (S) can be seen. A few sections further (b), the head of the larva is no longer visible. The syncytium (S) extends from the endodermis (E) at the edge of the cortex (C) through the vascular parenchyma, towards the xylem (X), and occupies an important part of the vascular cylinder. (c) Schematic representation of the infected root sample through which sections (a) and (b) were made. A potato cyst nematode (N) is feeding from a syncytium (S) induced inside a lateral root (R). Scale bars = 250 p.
not induced by G. tabacum, suggesting that feeding cells induced by root-knot and cyst nematodes might function more differently than originally thought. Arabidopsis thaliana as a model host Sijmons et aI.21 have recently shown that the small crucifer, A. thulium, is a suitable host for a number of plant-parasitic nematodes, including several Meloidogyne and Heteroderu species. Under optimized in vitro or greenhouse conditions, these nematodes complete their life cycle within A. thulium roots as in other host plants. These observations, combined with the advantages for molecular genetic analysis (small genome containing low amounts of repetitive DNA, amenability to transformation, a genome sequencing projecP), make A. thulium particularly well suited for the isolation of genes involved in plant-nematode interactions. Many A. thulium genes have been cloned and characterized. Their potential role in plan-nematode interactions may now be assessed by studying their expression upon infection. The isolation of the A. thulium gene encoding the protein kinase p34cdc2, a key regulator of the cell cycle*3,*4, has made possible the molecular study of the cell cycle in early phases of feeding cell formation. Using transgenic A. thulium plants containing a c&2 promoter-gus construct, induction of cdc2 was observed within one hour of initiation of feeding cells by cyst nematodes. c&2 expression was induced by both types of nematode for approximately three to four days, and then declined=. Similar results have recently been obtained with cycl, an A. thuliunu mitotic cyclin gene, indicating that mitosis was induced in both types of feeding cell (A. Niebel et al., unpublished). While DNA synthesis, and thus probably re-entry into the S phase
of the cell cycle, has been demonstrated in both types of feeding cells by tritiated thymidine experiments26J7, incomplete cell division had only been shown in rootknot nematode-induced giant cell@. Some unexpected common features thus seem to exist in the induction phase of both types of feeding cell. These results suggest that the early control of the cell cycle by root-knot and cyst nematodes in feeding cells could play an important role in their initiation. Some further similarities between feeding cells induced by root-knot or cyst nematodes and T. spirulis can be found. The animal parasite also induces nuclear hypertrophy and DNA synthesis, as recently demonstrated by incorporation of tritiated thymidine. In addition, T. spirufis was shown to arrest infected cells at the G2 /M phase of the cell cycle*s. Transgenic A. thulium lines containing promoter-gus fusions have also been used to analyze the expression of several other plant genes in H. schuchtii-induced syncytia. Most of the promoters tested showed no activity in syncytia*s. For instance, the patatin class I promoter from potato, which is activated in tissues functioning as nutritional sinks, is not expressed in syncytia, although M. incognita-induced giant cells are known to act as nutritional sinksao. A plasma membrane ATPase (Aha3) is also not expressed in syncytia. Studies on membrane potentials suggest, however, the presence of a proton-mediated co-transport of solutes into M. incognita-induced giant cells3*,3*. Although other genes involved in sink/source responses or in proton co-transport mechanisms might give different results, the absence of induction of Aha and Patatin I in syncytia may also be interpreted as a further illustration of the differences existing in the physiology of both types of feeding cell.
farasitology Today, vol. IO, no. I I, I994
Downregulation is al.so observed with several promoters in syncytia as compared to control root@. An example is phenylalanine ammonia lyase, the first enzyme in the phenylpropanoid pathway, which, among others, leads to the production of the defenserelated phytoalexins. This might once again suggest a local inhibition of defen.se mechanisms upon compatible nematode infection. Other molecular strategies, such as promoter tagging have been started. Based on the transformation and random integration of a pr’omoterless gus gene construct into A. fhdiunu, this method allows screening for tissuespecific or up-regulated promoters33. The elegance of the method lies in its ability to permit direct visualization of induced gus ex:pression in nematode feeding sites, at various stages of the interaction. The specificity of expression can simultaneously be studied by using uninfected parts of the same plant and control plants. In this way, three lines (553#2,553#25, 553#35), exhibiting relatively high levels of GUS staining inside H. s&a&ii-induced syncytia, have been isolated. In uninfected roots, GUS staining is restricted to the vascular cylinder29. Two other lines have recently been isolated. Arm1 (Arubidopsis-Meloidogyne 1) shows very high levels of GUS staining within M. incognita and H. s&a&ii-induced feeding cells, whereas staining in uninfected plants is restricted to the base of side roots and to some leaf veins. The second line expresses gus at the periphery of Hetenoderu-induced syncytia and in the vascular system of young leaves from infected and uninfected plants=. The isolation of the corresponding promoters bv inverse polymerase chain reaction should be rela&ely straightforward33. There are other reasons why A. thdiunu is an ideal model host: it has well-established genetics; it has a short life cycle (three months); and it is suitable for large-scale mutagenesis%. Important collections of mutants in several biosynthetic and developmental pathways have been generated in A. thuliunu and can be used to address specific questions. The influence of phytohormones, for example, which have been implicated in the formation of root-knot nematode feeding sites, has been assayed. Fourteen hormone mutants have been tested for their response to root-knot and cyst nematodes. Surprisingly, no significant differences in infection rates could be observed between mutants and wild-type plant@. These preliminary results require confirmation before any conclusions can be made about the role of phytohonnones in plant-rootknot or plant-cyst-nema.tode interactions. Currently, no root-knot or cyst-nematode-resistant ecotypes of A. thuliunu have been identified21,z. This was unexpected because bacteria- and fungi-resistant ecotypes had been isolated easily36. Arubidopsis thuliunu might be a poor host for sedentary nematodes in natural conditions (possibly because of its short life cycle, which would allow only limited reproduction of root-knot and cyst nematodes). Thus, co-evolution phenomena leading to gene-for-gene resistance mechanisms might not have taken place. A screen for mutagenesis-induced resistance to M. incognifu is being undertaken for A. fhuliunu. The aim is to identify plant genes important or essential for each stage of the complex interaction between root-knot nematodes and plants. A non-lethal mutation in one of these genes should lead to a reduction or even
427
Fig. 4. Electron micrograph ofa port ofo syncytium induced by the potato cyst nematode Globodera pallida in potato roots. Note the absence oftbe large central vacuole (which occupies most of the volume of a normal root cell), the dense cytoplasm (C), the abundance of mitochondria (M), the hypertrophied nucleus (N), and the cell wall ingrowths (CW/), which are thought to increase the nutrient uptake from phloem and @em elements (X). The connections between the different cells belonging to the same syncytium are not visible on this picture. Scale bar = I pm.
absence of infection symptoms. Twelve mutants have been isolated from approximately 15000 ethyl methane sulphonate-mutagenized plants by using two different screening procedure$J’. In vitro, these mutants show reductions of 50440% in numbers of galls per plant. For one of them, umi2, the resistance seems to be due to reduced penetration. Additional characterization is needed to determine the genetic background of these mutants as well as the stage of the interaction at which the mutation acts. Chromosome walking (map-based cloning) can then be initiated for the most interesting of them. A similar approach with H. schachtii on A. thuliunu has not yet yielded any stable mutants (F. Grundler, pers. commun.). Finally, the structural simplicity of the A. thuliunu root system%, and especially its translucent nature, has allowed spectacular progress. The behaviour of H. schuchtii and M. incognita larvae, especially during the first phases of their interaction with roots, has been observed using video-enhanced microscopy with a resolution and an accuracy never reached before with other host systems39,@. Furthermore, an approach using in vim microinjection directly into syncytia induced by cyst nematodes has recently been developed at the University of Kiel (Germany)Jl. This opens up a number of prospects, including syncytium manipulation using potential inhibitors and inducers, injection of nematode extracts in uninfected cells and the use of small amounts of feeding cell cytoplasm for PCR-based differential screening methods such as differential display”. Studies of incompatible interactions Resistance to root-knot and cyst nematodes exists in many crops@. It has often been found in related wild species and subsequently been introduced by breeding
Parasitology Today, vol. IO, no. I I, I 994
428
Two markers, gp79 and Apsl, have been mapped very close to the Mi resistance gene to Meloidogyne in tomatox,55. These markers might form a good starting point for chromosome walking towards the Mi gene, although the physical distances in that region seem greater than those predicted by linkage analysis5’j. Finally, a resistance gene to H. s&u&ii, which was introgressed from Beta proctrmbens into sugar beet (Beta vulgaris), has been mapped using addition lines57,58.
Stylet
Dorsal Gland Ampulla
Esophageal Lumen
Medium
Bulb
Dorsal Gland Extension
Subventral Extension Dorsal
Gland
Gland Secretory Granules
Subventral Glands Intestine
Fig.5. Anterior part ofo plant-parasitic nematode in itsj2 stage (the infkctive stage). Morphological adaptations to parasitic behaviour (such as the presence of a stylet used to pierce plant tissues as well as to inject or retrieve fluids into and from feeding cells) can be seen. Also important for parasitic behaviour are the three oesophageal glands (two subventral and one dorsal). They are thought to play an important role especially during feeding cell induction and maintenance. (Adapted, with permission, from Ref: 64.) into commercial varieties. The resistance reactions toward root-knot and cyst nematodes, which are often hypersensitive 44, have been extensively characterized morphologically and ultrastructurally45-49. However, the product(s) of the resistance gene(s), as well as the exact mechanism by which they induce resistance, remain unknown. Hammond-Kosack et ~I.50analyzed two-dimensional protein electrophoresis patterns after in vitro translation of mRNA species isolated from potatoes that were either uninfected or infected by virulent and avirulent G. rostochiensis pathotypes. After infection, considerable changes could be seen systemically in leaves, but only some minimal effects were detected in roots. However, effects specific for the resistance reaction in roots were observed. A more direct approach to identify the product(s) of nematode resistance genes is to clone these genes by mapping and chromosome-walking approaches. Several monogenic resistance traits have recently been mapped. Linkage analysis has been used to map two resistance genes to G. rostochiensis in potato. Grol, derived from Solanum speguzzini, was mapped to potato linkage group IX (Ref. 51), and HZ, derived from S. tuberosum ssp andigenu, to chromosome 5 (Refs 52,53).
Developing new resistant varieties Chemical nematicides, widely used in the past, will probably gradually loose their importance in the coming years. First, their toxicity and poor biodegradability in soils make them incompatible with a modern, environmentally conscious approach to agriculture. Second, the cost of repeated nematicidal treatments is high and essentially unaffordable for Third World countries. The use of resistant varieties forms a useful alternative and should be developed further as a primary control option. Classical breeding has allowed the selection of several resistant varieties. However, for some crops, resistant varieties do not exist, while for most others resistance against only some species or pathotypes is found. Some of the commercially used resistance traits are temperature sensitive, and therefore unsuited for use in tropical and subtropical region@. More breeding efforts will be needed to achieve a satisfactory level of protection. On the other hand, the introduction of resistance traits from related wild species into commercial varieties by classical breeding is very time consuming. It is also usually difficult to separate the resistance trait from undesirable and agronomically unacceptable factors, linked to the resistance gene. The cloning of monogenie resistance genes and their transfer to other plant varieties or species will circumvent these problems and provide farmers with new resistant cultivars. It should also become possible, especially if information about the function of these genes is available, to engineer novel types of resistance (eg. with broader host ranges, or reduced temperature sensitivity) by molecular manipulations of the cloned resistance genes. It should be mentioned that monogenic resistance traits, with gene-for-gene relationships, are usually unstable under conditions of high selective pressurem. Other strategies, based on the study of plant genes induced during a compatible interaction, could lead to more stable resistances. The idea is to use promoters that are up-regulated in feeding cells to express proteins that would kill or harm the nematode, or the feeding cell on which the parasite depends. Currently, promoters expressed exclusively in feeding cells have not been identified and are not very likely to exist, for evolutionary reasons. To counterbalance the possible ‘leakiness’ of promoters induced in feeding cells but also expressed in different tissues or during specific phases of development, a two-component system has been designed61@. In such a system, the promoter of a gene expressed in nematode-induced feeding cells is fused to a ‘killer gene’ (such as an RNase). This construct is then transferred to plants, constitutively expressing a gene that would neutralize the effect of the ‘killer gene’ outside of the nematode feeding site. Preferably, the constitutive
Parasitology
Today,vol. IO, no. I I, I 994
promoter used should be down-regulated in feeding cells. Such down-regulation of several constitutive promoters has been observed inside syncytia29. A typical example of a two-component system, which has been used to engineer male-sterile plants, is the RNase barnase, coupled to its specific inhibitor barstafi3. Another possibility is to identify and isolate strictly nematode-responsive elements within nematode-upregulated promoters20 and to use these to drive the expression of a ‘killer gene’. The parasite’s point of -view Plant-parasitic nematodes have evolved morphological adaptations to parasitism, including a stylet (which allows them to pierce host tissues and to inject or withdraw fluids from plant cells) and highly specialized oesophageal glands (F$. 5). For both root-knot and cyst nematodes, a role for stylet secretions (saliva) in induction and maintenance of giant cells or syncytia has been suggested (for a review, see Ref. 64). Secretory granules from the dorsal gland, an organ that enlargens and becomes more active after the onset of the parasitic phase of the larva (Fig. 5), could play a crucial role65,M. Studies on nematode secretions have revealed the presence of at least nine proteins in stylet exudate of excised Meloidogyne adult female@. Monoclonal antibodies (mAbs) raised against isolated secretory granules from the dorsal gland of Meloidogyne have allowed the immunopurification of a 212 kDa glycoprotein. The exact function of this protein is still unknowr@. Monoclonal antibodies have been raised against specific structures of root-knot and cyst nematodes and larval stage-specific mAbs have been identifiedeg-71. A mAb specific for the amphids (an organ involved in chemoreception) of various Mebidogyne species” partly impairs the ability of nematodes to locate roots”. Besides their fundament,al interest for the understanding of the plant-nematode interactions, some mAbs could be used to engineer resistance via the ‘plantibody’ technology 74. The lidea is to express mammalian mAbs directed against precise antigens of the nematode within plants. Hiatt et al.75 first expressed whole antibodies in plants. Functional single-chain antibodies (ScAbs), which have fewer requirements for assembly, can be expressed to high levels in the apoplasm via the default secretion pathway76. This is encouraging for the engjneering of nematode resistance using mAbs, such as the amphid-specific mAbs, that could interfere with migrating larvae. Expression of antibodies in the cytosol leads to much lower ScAb accumulation, probably because of instability of the antibody76,n. However, this is still enough to cause delayed and reduced .virus infection symptoms78. Plants expressing mAbs directed against oesophageal gland secretions, which are possibly injected into the cytoplasm of future feeding cells, might therefore show some protection ag:ainst nematodes. Conclusions The interaction between plants and root-knot and / or cyst nematodes is studied intensively for both fundamental and applied reasons. The recent introduction of recombinant DNA and protein techniques into plant nematology, as well as the use of the model host system, A. fhakzna, has opened new perspectives. On the plant side, genes expressed in roots upon infection have
429 been isolated and a picture of the complex molecular changes induced by nematodes has started to emerge. In addition, resistance genes have been mapped and their cloning by chromosome walking has been initiated. On the nematode side, some of the components thought to be involved in the induction of feeding cells have been isolated and studied. The approaches discussed in this paper are only beginning to yield valuable data and much remains to be understood about this fascinating plant-parasite interaction. However, these approaches have already contributed to the evolution of plant nematology from a more descriptive towards a more analytical phase. Acknowledgements The authors thank Tom Gerats and Jonathan Clarke for critical reading, Mat-tine De Cock for help with the manuscript, and Vera Vetmaercke and Karel Spruyt for the figures. This work was supported by a grant from the Vlaams Actieprogramma Biotechnologie (No. 073). GG is a Senior Research Assistant ofthe National Fund for Scientific Research (Belgium). References 1 Sasser, J.N. and Freckman, D.W. (1987) in Vistas on Nematology: A Commemoration of the Twenty-Ffth Anniversary of the Society of Nemafologists (Veech, J.A. and Dickson, D.W., eds), pp 7-14, Society of Nematologists Inc. 2 Despommier, D.D. (1990) PurusitoIogy Today 6,193-196 3 Bird, A.F. (1961) 1. Biophys. Biochem. Cytol. 11,701-715 4 Endo, B.Y. (1971) in Plant Parasitic Nematodes (Vol. II) (Zuckerman, B.M., Mai, W.F. and Rohde, R.A., eds), pp 91-117, Academic Press 5 Jones, M.G.K. and Dropkin, V.H. (1975) Physiol. Plant Puthol. 5, 119-124 6 Pate, J.S. and Gunning, B.E.S. (1972) Annu. Rev. Plant Physiol. 23, 173-196 7 Jones, M.G.K. and Northcote, D.H. (1972) J. CeZZSci. 10,789-809 8 Huang, C.S. and Maggenti, A.R. (1969) Phytopathology 59, 447-455 9 Bird, A.F. (1973) Physiol. Plant PathoZ. 3,387-391 10 Jones, M.G.K. and Payne, H.L. (1978) 1. NematoZ. 10,70-84 11 Huang, C.S. (1985) in An Advanced Treatise on Meloidogyne, VoZ.I: Biology and Control (Sasser, J.N. and Carter, C.C., eds), pp 155-164, Department of Plant Pathology and US Agency for International Development 12 Gurr, S.J. et al. (1991) Mol. Gen. Genef. 226,361-366 13 Niebel, A. et al. (1993) Arch. Int. Physiol. Biochim. Biophys. 101, B18 14 Lamb, C.J. (1994) Cell 76,419-422 15 Chen Z., Silva, H. and KIessig, D.F. (1993) Science 262, 1883 -1886 16 Niebel, A. et al. (1993) PZant Cell 5,1697-1710 17 Carpita, N.C. and Gibeaut, D.M. (1993) Plant ].3,1-30 18 Ye, Z-H. and Vamer, J.E. (1991) Plant Cell 3,23-37 19 Esquerr&Tugay& M.T. et al. (1979) PZunf Physiol. 64,320-326 20 Opperman, C.H., Taylor, C.G. and Conkling, M.A. (1994) Science 263,221-223 21 Sijmons, P.C. et al. (1991) Plant ].1,245-254 22 Somerville, C. (1989) Plant CeZZ1,1131-1135 23 Ferreira, P.C.G: et aZ: (1991) Plant CeZZ3,531-540 24 Hemerlv. A.S. et al. (1993) Plant Cell 5.1711-1723 25 Niebel,‘i. et al. h A&nces in kIoZecuZar Plant Nemafology (NATO-AS1 Series) (Lamberti, F., De Giorgi, C. and McBird, D., eds), Plenum Press (in press) 26 Rubinstein, J.H. and Owens, R.G. (1964) Confrib. Boyce Thompson Inst. 22,491-502 27 Endo, B.Y. (1971) Phytopathology 61,395-399 28 Jasmer, D.P. (1993) J. Cell BioZ. 121,785-793 29 Goddijn, O.J.M. et al. (1993) Plant J. 4,863-873 30 Bird, A.F. and Loveys, B.R. (1975) 1. NematoZ. 7,111-113 31 Jones, M.G.K. et al. (1974) Protoplasma 80,401-405 32 Jones, M.G.K. (1981) Ann. AppZ. BioZ. 97,353-372 33 Lindsev, K. et al. (1993) Trunsaenic Res. 2,33-47 34 Meyer&vitz, E.M: (1989) CeZZ>, 263-269 35 Siimons. P.C.. von Mende. N. and Grundler. F.M.W. in Aiabiddpsis (Somenrille, C.’ and Meyerowitz, E., eds), Cold Spring Harbor Laboratory Press (in press)
PorositologyToday, vol. IO, no. I I, I994
430 36 Dangl, J.L. et al. (1992) in Methods in Arabidopsis Research (Koncz, C., Chua, N-H. and ScheII, J., eds), pp 393-418, World Scientific 37 Niebel, A. et al. (1993) in Mechanisms of Plant Defense Responses (Development in Plant PuthoZogy, Vol. 2) (Fritig, B. and Legrand, M., eds), pp __ 344-348, Kluwer Academic Publishers 38 Dolan, L. et al. (1993) Development 119,71-84 39 Wvss. U. and Gnmdler. F.M.W. (19921 Nematologica 38.488-493 40 W&s) U., Grundler, F.M.W. ani M&h, A. (1932) N&natologica 38,98-111 41 BBckenhoff, A. and Grundler, F.M.W. Parasitology (in press) 42 Liane. I?. and Pardee. A.B. (1992) Science 257,967-971 43 Sidh;, G.S. and Webster, J.&I. (1481) Bat. Rev. 47,387-419 44 Robinson, M.P., Atkinson, H.J. and Perry, R.N. (1988) Rev. NtfmatoI. 11,99-107 45 Kim, Y.H., Riggs, R.D. andKim, K.S. (1987)J. Netnatol. 19,177-187 46 Rice, S.L., Stone, A.R. and Leadbeater, B.S.C. (1987) Physiol. Mol. Plant Pathol. 31, l-14 47 Bleve-Zacheo, T., MeliIlo, M. and Zacheo, G. (1990) Rev. Nt+natol. 13, 29-36 48 Endo, B.Y. (1991) Rev. Ntfmutol. 14,73-94 49 Golinowksi, W. and Magnusson, C. (1991) Can. J. Bat. 69,53-62 50 Hammond-Kosack, K.E., Atkinson, H.J. and Bowles, D.J. (1990) Physiol. Mol. Plant PathoI. 37‘339-354 51 Barone, A. et al. (1990) Mol. Gen. Genet. 224,177-182 52 Pineda, O., Bonierdale, M.W. and Plaisted, R.L. (1993) Genome 36,152-156 53 Gebhardt, C. et al. (1993) Theor. Appl. Genet. 85,541-544 54 Messeguer, R. et al. (1991) Theor. Appl. Genet. 82,529-536 55 Ho, J-Y. et al. (1992) Plant J. 2,971-982
56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78
van Daelen, R.A.J.J. et al. (1993) Plant Mol. Biol. 23, 185-192 Jung, C. et al. (1992) Mol. Gen. Genet. 232,271-278 Salentijn, E.M.J. et al. (1992) MoI. Gen. Genet. 235,432~440 Roberts, P.A. (1992) 1. Nematol. 24,213-227 Jarquin-Barberena, H. et UI.(1991) Rev. N&nutoZ. 14,261-275 Sijmons, I’. et al. (1993) Patent Application No. PCT/EP92/02559, WO 93/ 10251 Van der Eycken, W. et al. (1992) Patent Coop. Treaty EP/92/01214, Int. Publ. No. WO 92 /21757 Mariani, C. et al. (1992) Nature 357,384-387 Hussey, R.S. (1989) Annu. Rev. Phytupathol. 27,123-141 Endo, B.Y. (1987) 1. Nematol. 19,469-483 Hussey, R.S. and Mims, C.W. (1990) ProtopIusmu 156,9-18 Veech, J.A., Starr, J.L. and Nordgren, R.M. (1987) 1. Nemutol. 19, 463-468 Hussey, R.S., Paguio, O.R. and Seabury, F. (1990) Phytopathology 80,709-714 Atkinson, H.J. and Harris, P.D. (1989) Parasitology 98,479-487 Davis, E.L. et uI. (1992) Phytoputhology 82,1244-1250 Backett, K.D., Atkinson, H.J. and Forrest, J.M.S. (1993) J. Nemutol. 25,395-400 Stewart, G.R., Perry, R.N. and Wright, D.J. (1993) Parasitology 107,573-578 Stewart, G.R. et al. (1993) Parasitology 106,405-412 Schots, A. et al. (1992) Neth. J. Plant Pathol. 98 (Suppl. 2), 183-191 Hiatt, A., Cafferkey, R. and Bowdish, K. (1989) Nature 342,76-78 Firek, S. et al. (1993) Plant Mol. Biol. 23,861-870 Owen, M. et al. (1992) BiolTechno2ogy 10,790-794 Tavladoraki, I’. et al. (1993) Nature 366,469-472
The Role of Salivary Vasodilators in Bloodfeeding and Parasite Transmission D.E. Champagne In this paper, Donald Champagne reviews the salivary vasodilators, points to effects of similar compounds that may be shared by the insect substances, and discusses the potential significance of these effects with regard to parasite transmission. Bloodfeeding is a complex task made difficult by the presence of hemostatic mechanisms used by vertebrate hosts to prevent blood 1ossQ. In the case of small lesions, such as those produced by a feeding arthropod, the most significant components of hemostasis are: (1) the formation of a platelet plug (in response to ADP and collagen); and (2) vasoconstriction. Although the hematophagous habit has arisen independently many times, all arthropods that have been examined to date use the enzyme apyrase to inhibit platelet aggregation by hydrolysing ADP and ATPQ. In contrast, a remarkable diversity of substances have been employed as vasodilators (for summary see Table 1). In several instances, these substances are close mimics of, or identical to, endogenously produced signal molecules that affect systems other than vasodilation, including components of the immune response.
Donald Champagne is at the Department of Entomology, University of Arizona, Tucson, AZ 8572 I, USA.
Mosquitoes The salivary vasodilator of the yellow fever vector Aedes aegypti, first noted by Pappas et a1.3, was subsequently characterized as a tachykinin based on its endothelium dependence, cross-desensitization with the mammalian tachykinin substance P and reaction with an anti-substance I.’antibodyd. When the vasodilator was purified and sequenced, it was shown to comprise two novel tachykinins, sialokinin I and II, the first tachykinins to be isolated from an insects. Structurally, these peptides combine characteristics of substance P with those typical of neurokinins, the second major subgroup of mammalian tachykinins. These peptides were as potent as substance P in assays on the guinea-pig ileum. The Lacrosse Fever vector, Aedes triseriatus, also employs a tachykinin, which differs from the sialokinins in its chromatographic properties6. Indeed, the use of vasodilatory tachykinins may be characteristic of the genus Aedes. Substance P is the most abundant tachykinin in skin, where its primary role is as a sensory neuromodulator. In addition, substance P and other tachykinins including the neurokinins stimulate the oxidative burst and thromboxane release from macrophages7JJ. Of particular interest, both neurokinin A and (to a lesser extent) substance P enhance the primary antibody response in macrophages: maximum effect was seen at 10~~ but responses were produced by 0
1994, Elsev,er Sc,ence Ltd
424
Plant-Cyst Nematode and Plant-Root-knot Nematode Interactions A. Niebel, G. Gheysen and M. Van Montagu Root-knot nematodes and cyst nematodes are obligate plant parasites that cause extensive damage to the agriculture of both temperate and tropical countries. In this review, Andreas Niebel, Godelieve Gheysen and Marc Van Montagu describe how, in the past decade, the use of molecular techniques has provided new insights in the complex interactions between these sedentary plant-parasitic nematodes and their infected host plants. They give an account of the progress in OUY understanding of both the parasite and the host during compatible and incompatible interactions. They also outline the importance of a new model host system, Arabidopsis thaliana. Among plant-parasitic nematodes, the root-knot nematodes (Meloidogyninae) and the cyst nematodes (Heteroderinae) have, over the past decade, received increasing attention. This is largely a consequence of the agronomic impact of one root-knot nematode genus (Meloidogyne) and two cyst nematode genera (Heterodera and Globoderu). Meloidogyne is the most destructive plant-parasitic nematode genus in the world. It infects over 2000 plant species, contributing greatly to an estimated annual yield loss of US$77 billionl. Although Meloidogyne is nearly ubiquitous, it represents a particular threat to agriculture in tropical and sub-tropical regions. In contrast, Heteroderu and Globoderu are the greatest problem for agriculture in more temperate regions. Both types of plant-parasitic nematodes are obligate parasites, spending the major part of their life cycle within roots (Figs 1 and 2). In common with some animal parasites, such as Trichinella spirulis2, they do not kill the host cells from which they feed. Instead, the nematodes induce a redifferentiation process that leads to the formation of multinucleated feeding cells on which they depend for the completion of their life cycle (Figs l-3). These feeding cells appear to be metabolically active: their cytoplasm is very dense, containing numerous mitochondria, plastids, ribosomes, welldeveloped Golgi apparatus, and smooth endoplasmic reticulum, generally organized in swirls. The central vacuole disappears and gives rise to many small vacuoles; nuclei and nucleoli become hypertrophic and assume an amoeboid profilea-5. In addition, special secondary cell wall formations develop (Fig. 4). These are called ‘cell wall ingrowths’ and are typical of transfer cell+. Cell wall ingrowths are thought to enhance solute uptake from the vascular system7. Parallels can be drawn with T. spiralis, where a plexus of venules, called placenta, surrounds the parasitized muscle cells, probably to facilitate transport between the parasite and its hostz. Although feeding cells Andreas Niebel, Godelieve Gheysen and Marc Van Montagu are at the Labotatotium voor Genetica, Univeniteit Gent B-9000 Gent, Belgium.
induced by root-knot and cyst nematodes are similar in ultrastructure and possibly in function, they are thought to be formed by distinct mechanisms. Cyst nematodes have been shown to induce a single, multinucleated syncytium by cell wall breakdown. Despite a long controversy gs, it is now generally accepted that cell wall breakdown plays no role in the formation of root-knot nematode-induced feeding cells. In this case, the parasite induces several ‘giant cells’ by repeated mitosis without CytokinesisiQli. Studies of compatible plant-nematode interactions Although the descriptive knowledge of plant-nematode interactions is extensive, little is known about the underlying biochemical and molecular events leading to the observed redifferentiation processes. Some years ago, several molecular approaches were initiated to identify and characterize plant genes altered in expression after infection by root-knot or cyst nematodes. The main problem in analyzing gene expression at nematode feeding sites is the limited number of root cells affected by the nematode. To circumvent this problem, Gurr et al.12 developed a polymerase chain reaction @‘CR)-based method that allowed them to construct a cDNA library from small amounts of Globoderu-infected potato root sectors (at the adult stage of the parasite, 26 days after inoculation). They then differentially screened the library with probes derived from infected and uninfected roots. One differentially expressed clone, pl’MR1, was induced locally within four days of inoculation. It showed no sequence homology with known sequences. To understand the role of pPMR1 in the plant-nematode interaction, further work is required, specifically, detailed expression analysis and precise cellular localization of the transcript. In our laboratory, a cDNA library was constructed using G. pallidu-infected potato roots (at the adult stage of the parasite) and differentially screened. Several G. pallida-induced potato genes were identifiedis. Sequence identity indicates that one of these, Nem2, encodes a potato catalase. Expression analysis showed that Nem2 is gradually induced after Globoderu attack and reaches its strongest expression in roots and stems of infected plants, approximately four weeks after inoculation. Other nematodes (M. incognita), as well as bacterial root pathogens (Enuiniu curotovoru and Coynebacterium sepedonicum), also induce this catalase in potato roots and stems. Catalase is a peroxisomal heme protein catalyzing the dismutation of hydrogen peroxide into water and oxygen. Hydrogen peroxide functions as a defense-related signal transducer, triggering plant defense mechanism+. In addition, the inhibition of catalase by salicylate has been shown to lead to a hydrogen peroxide-mediated systemic acquired resistance phenomenonis. We believe that compatible root pathogens in general, 0
1994, Elsev~er Swnce
Ltd
Porasiro/ogy
Today, vol.
IO, no. I I, I994
425
Fig. 1. Life cycle of a cyst nematode. (I) The larvae of the second stage (a) are attracted to the roots. After penetration, they migrate intmcellulorfy, direct/y toward the vascular cylinder. There, they start feeding upon a sing/e cell, which is rapid/y turned into a large syncytium by incorporation of neighbouring tissue (through cell wall breakdown). (2) The larvae undergo three additional moults.Here o female (b) and a mole (c) of the fourth larvul stage are shown. Whereas the body of the larvae bulges out of the root, their head remains embedded witlllin the root for food withdrawal from the syncytium. (3) During the last mou/t, the ma/e (e) changes shape dmm&a//y, /eaves the root, fenti/izes the female (d), and then dies. (4) At moturity, the cuticle of the fimole hardens and turns into a cyst (f) containing the eggs. Inside the eggs, the]/ larvae undergo their first moulc The 12 larvae (a) then hatch, often under the influence of specific substances present in root exudates. Depending on environmental conditions,the cycleis completed in one to two months.
and nematodes in particular, might have evolved mechanisms to increase plant catalase levels, thereby inhibiting or retarding hydrogen peroxide-mediated defense reactions. The role of the structural cell wall protein, extensin, during nematode infecuon has been studied in detail in tobacco*6. Extensin, one of the most abundant structural cell wall proteins, is thought to play a role in the termination of cell expansion and, thus, in cell wall rigidity, by being crosslinked either to itself or to other cell wall components*7. Extensin is induced in actively dividing cells (where the synthesis of new cell walls requires new structural material)ls, following compatible and incompatible pathogen attack19 and also several abiotic stresses. The tobacco cyst nematode, G. tubacum, only weakly induces extensin expression, probably due to wounding during penetration and migration through the roots. High extensin expression is observed in galls induced by M. juvanicu, especially during the second larval stage. The highest extensin expression can be found in the gall cortex. These cells may need to increase the rigidity of their cell walls in order to resist the mechanical pressure created by dividing pericycle cells (that also show significant extensin expression) and growing giant cells and nematodes. No significant amounts of extensin could
be found in the large cell walls of giant cells and particularly not in the cell wall ingrowths. This may illustrate the need for a loose cell wall structure (achieved in the absence of a dense protein network) for efficient solute uptake from the vascular system. It also indicates the ‘extensible’ state of the cell walls of expanding giant cells. A member of a membrane protein family, pRB7, believed to function as water channels, has recently been shown to be up-regulated in giant cells induced by different species of Meloidogyne2’J. Using transgenic plants containing the fobRB7 (the gene encoding pRB7) promoter fused to the coding sequence of the j3-glucuronidase A (gus) reporter gene, the authors could show that tobRB7 is transcribed in and around giant cells from four days until 40 days after inoculation. The expression in control roots is restricted to root me&terns and immature vascular cylinder regions. Promoter deletion studies showed that the root-specific expression pattern could be uncoupled from the nematode-induced gene expression. A 299bp S-flanking region was shown to confer giant cellspecific expression. The pRB7 protein might be important in regulating the water status of giant cells once their complex secondary cell wall and membrane modifications have developed. Interestingly, tobRB7 is
Fig. 2. Life cycle of o root-knot nematode. (I) The larvae of the second stage (0) are attracted to the roots. They usually penetrote the roots closely behind the root tip. The larvae then migrate first towards the root tip, where the absence ofdiferentiated endodermis allows them to enter the vascular cylinder. This migration happens intercellular/y by mechanical and possibly enzymatic sofiening of the middle lame/la. The parasites finaIry start feeding on three to ten cells, which are rapidly turned into mu/tinucleated giant cells, by endomitosis and cell hypertrophy. (2) At the same time as the giant cells are firmed, the ce//.sof the neighbouring pericyc/e start to divide, giving rise to a typica/ go// or root-knot Inside the gal/, D female (b) and o mole (c) of the 13 larval stage ore shown. (3) The gall continues to swell, while females (d) and moles (e) ore in their j4 stage. (4) During the last mou/t, the ma/e (h) dmmoticolly changes its shape, then leaves the root and fertilizes the female (f) in the case ofomphimictic species, However, parthenogenesis is often encountered in root-knot nemutodes. The female lc~ysits eggs in a gelatinous matrix (g) outside the root From there, the larvae of the second stage (a) hatch and are ottmcted to roots. Depending on environmental conditions, this cyc/e is completed in one to two months.
Parasitology Today, vol. IO, no. I I, I 994
426
b Fig. 3. Light micrograph of a cross-section through a potato root infected by the potato cyst nematode Globodera pallida. (a) The head of the nematode (N) inserted into the root is clearly visible, whereas only the distal part of the induced syncytium (S) can be seen. A few sections further (b), the head of the larva is no longer visible. The syncytium (S) extends from the endodermis (E) at the edge of the cortex (C) through the vascular parenchyma, towards the xylem (X), and occupies an important part of the vascular cylinder. (c) Schematic representation of the infected root sample through which sections (a) and (b) were made. A potato cyst nematode (N) is feeding from a syncytium (S) induced inside a lateral root (R). Scale bars = 250 p.
not induced by G. tabacum, suggesting that feeding cells induced by root-knot and cyst nematodes might function more differently than originally thought. Arabidopsis thaliana as a model host Sijmons et aI.21 have recently shown that the small crucifer, A. thulium, is a suitable host for a number of plant-parasitic nematodes, including several Meloidogyne and Heteroderu species. Under optimized in vitro or greenhouse conditions, these nematodes complete their life cycle within A. thulium roots as in other host plants. These observations, combined with the advantages for molecular genetic analysis (small genome containing low amounts of repetitive DNA, amenability to transformation, a genome sequencing projecP), make A. thulium particularly well suited for the isolation of genes involved in plant-nematode interactions. Many A. thulium genes have been cloned and characterized. Their potential role in plan-nematode interactions may now be assessed by studying their expression upon infection. The isolation of the A. thulium gene encoding the protein kinase p34cdc2, a key regulator of the cell cycle*3,*4, has made possible the molecular study of the cell cycle in early phases of feeding cell formation. Using transgenic A. thulium plants containing a c&2 promoter-gus construct, induction of cdc2 was observed within one hour of initiation of feeding cells by cyst nematodes. c&2 expression was induced by both types of nematode for approximately three to four days, and then declined=. Similar results have recently been obtained with cycl, an A. thuliunu mitotic cyclin gene, indicating that mitosis was induced in both types of feeding cell (A. Niebel et al., unpublished). While DNA synthesis, and thus probably re-entry into the S phase
of the cell cycle, has been demonstrated in both types of feeding cells by tritiated thymidine experiments26J7, incomplete cell division had only been shown in rootknot nematode-induced giant cell@. Some unexpected common features thus seem to exist in the induction phase of both types of feeding cell. These results suggest that the early control of the cell cycle by root-knot and cyst nematodes in feeding cells could play an important role in their initiation. Some further similarities between feeding cells induced by root-knot or cyst nematodes and T. spirulis can be found. The animal parasite also induces nuclear hypertrophy and DNA synthesis, as recently demonstrated by incorporation of tritiated thymidine. In addition, T. spirufis was shown to arrest infected cells at the G2 /M phase of the cell cycle*s. Transgenic A. thulium lines containing promoter-gus fusions have also been used to analyze the expression of several other plant genes in H. schuchtii-induced syncytia. Most of the promoters tested showed no activity in syncytia*s. For instance, the patatin class I promoter from potato, which is activated in tissues functioning as nutritional sinks, is not expressed in syncytia, although M. incognita-induced giant cells are known to act as nutritional sinksao. A plasma membrane ATPase (Aha3) is also not expressed in syncytia. Studies on membrane potentials suggest, however, the presence of a proton-mediated co-transport of solutes into M. incognita-induced giant cells3*,3*. Although other genes involved in sink/source responses or in proton co-transport mechanisms might give different results, the absence of induction of Aha and Patatin I in syncytia may also be interpreted as a further illustration of the differences existing in the physiology of both types of feeding cell.
farasitology Today, vol. IO, no. I I, I994
Downregulation is al.so observed with several promoters in syncytia as compared to control root@. An example is phenylalanine ammonia lyase, the first enzyme in the phenylpropanoid pathway, which, among others, leads to the production of the defenserelated phytoalexins. This might once again suggest a local inhibition of defen.se mechanisms upon compatible nematode infection. Other molecular strategies, such as promoter tagging have been started. Based on the transformation and random integration of a pr’omoterless gus gene construct into A. fhdiunu, this method allows screening for tissuespecific or up-regulated promoters33. The elegance of the method lies in its ability to permit direct visualization of induced gus ex:pression in nematode feeding sites, at various stages of the interaction. The specificity of expression can simultaneously be studied by using uninfected parts of the same plant and control plants. In this way, three lines (553#2,553#25, 553#35), exhibiting relatively high levels of GUS staining inside H. s&a&ii-induced syncytia, have been isolated. In uninfected roots, GUS staining is restricted to the vascular cylinder29. Two other lines have recently been isolated. Arm1 (Arubidopsis-Meloidogyne 1) shows very high levels of GUS staining within M. incognita and H. s&a&ii-induced feeding cells, whereas staining in uninfected plants is restricted to the base of side roots and to some leaf veins. The second line expresses gus at the periphery of Hetenoderu-induced syncytia and in the vascular system of young leaves from infected and uninfected plants=. The isolation of the corresponding promoters bv inverse polymerase chain reaction should be rela&ely straightforward33. There are other reasons why A. thdiunu is an ideal model host: it has well-established genetics; it has a short life cycle (three months); and it is suitable for large-scale mutagenesis%. Important collections of mutants in several biosynthetic and developmental pathways have been generated in A. thuliunu and can be used to address specific questions. The influence of phytohormones, for example, which have been implicated in the formation of root-knot nematode feeding sites, has been assayed. Fourteen hormone mutants have been tested for their response to root-knot and cyst nematodes. Surprisingly, no significant differences in infection rates could be observed between mutants and wild-type plant@. These preliminary results require confirmation before any conclusions can be made about the role of phytohonnones in plant-rootknot or plant-cyst-nema.tode interactions. Currently, no root-knot or cyst-nematode-resistant ecotypes of A. thuliunu have been identified21,z. This was unexpected because bacteria- and fungi-resistant ecotypes had been isolated easily36. Arubidopsis thuliunu might be a poor host for sedentary nematodes in natural conditions (possibly because of its short life cycle, which would allow only limited reproduction of root-knot and cyst nematodes). Thus, co-evolution phenomena leading to gene-for-gene resistance mechanisms might not have taken place. A screen for mutagenesis-induced resistance to M. incognifu is being undertaken for A. fhuliunu. The aim is to identify plant genes important or essential for each stage of the complex interaction between root-knot nematodes and plants. A non-lethal mutation in one of these genes should lead to a reduction or even
427
Fig. 4. Electron micrograph ofa port ofo syncytium induced by the potato cyst nematode Globodera pallida in potato roots. Note the absence oftbe large central vacuole (which occupies most of the volume of a normal root cell), the dense cytoplasm (C), the abundance of mitochondria (M), the hypertrophied nucleus (N), and the cell wall ingrowths (CW/), which are thought to increase the nutrient uptake from phloem and @em elements (X). The connections between the different cells belonging to the same syncytium are not visible on this picture. Scale bar = I pm.
absence of infection symptoms. Twelve mutants have been isolated from approximately 15000 ethyl methane sulphonate-mutagenized plants by using two different screening procedure$J’. In vitro, these mutants show reductions of 50440% in numbers of galls per plant. For one of them, umi2, the resistance seems to be due to reduced penetration. Additional characterization is needed to determine the genetic background of these mutants as well as the stage of the interaction at which the mutation acts. Chromosome walking (map-based cloning) can then be initiated for the most interesting of them. A similar approach with H. schachtii on A. thuliunu has not yet yielded any stable mutants (F. Grundler, pers. commun.). Finally, the structural simplicity of the A. thuliunu root system%, and especially its translucent nature, has allowed spectacular progress. The behaviour of H. schuchtii and M. incognita larvae, especially during the first phases of their interaction with roots, has been observed using video-enhanced microscopy with a resolution and an accuracy never reached before with other host systems39,@. Furthermore, an approach using in vim microinjection directly into syncytia induced by cyst nematodes has recently been developed at the University of Kiel (Germany)Jl. This opens up a number of prospects, including syncytium manipulation using potential inhibitors and inducers, injection of nematode extracts in uninfected cells and the use of small amounts of feeding cell cytoplasm for PCR-based differential screening methods such as differential display”. Studies of incompatible interactions Resistance to root-knot and cyst nematodes exists in many crops@. It has often been found in related wild species and subsequently been introduced by breeding
Parasitology Today, vol. IO, no. I I, I 994
428
Two markers, gp79 and Apsl, have been mapped very close to the Mi resistance gene to Meloidogyne in tomatox,55. These markers might form a good starting point for chromosome walking towards the Mi gene, although the physical distances in that region seem greater than those predicted by linkage analysis5’j. Finally, a resistance gene to H. s&u&ii, which was introgressed from Beta proctrmbens into sugar beet (Beta vulgaris), has been mapped using addition lines57,58.
Stylet
Dorsal Gland Ampulla
Esophageal Lumen
Medium
Bulb
Dorsal Gland Extension
Subventral Extension Dorsal
Gland
Gland Secretory Granules
Subventral Glands Intestine
Fig.5. Anterior part ofo plant-parasitic nematode in itsj2 stage (the infkctive stage). Morphological adaptations to parasitic behaviour (such as the presence of a stylet used to pierce plant tissues as well as to inject or retrieve fluids into and from feeding cells) can be seen. Also important for parasitic behaviour are the three oesophageal glands (two subventral and one dorsal). They are thought to play an important role especially during feeding cell induction and maintenance. (Adapted, with permission, from Ref: 64.) into commercial varieties. The resistance reactions toward root-knot and cyst nematodes, which are often hypersensitive 44, have been extensively characterized morphologically and ultrastructurally45-49. However, the product(s) of the resistance gene(s), as well as the exact mechanism by which they induce resistance, remain unknown. Hammond-Kosack et ~I.50analyzed two-dimensional protein electrophoresis patterns after in vitro translation of mRNA species isolated from potatoes that were either uninfected or infected by virulent and avirulent G. rostochiensis pathotypes. After infection, considerable changes could be seen systemically in leaves, but only some minimal effects were detected in roots. However, effects specific for the resistance reaction in roots were observed. A more direct approach to identify the product(s) of nematode resistance genes is to clone these genes by mapping and chromosome-walking approaches. Several monogenic resistance traits have recently been mapped. Linkage analysis has been used to map two resistance genes to G. rostochiensis in potato. Grol, derived from Solanum speguzzini, was mapped to potato linkage group IX (Ref. 51), and HZ, derived from S. tuberosum ssp andigenu, to chromosome 5 (Refs 52,53).
Developing new resistant varieties Chemical nematicides, widely used in the past, will probably gradually loose their importance in the coming years. First, their toxicity and poor biodegradability in soils make them incompatible with a modern, environmentally conscious approach to agriculture. Second, the cost of repeated nematicidal treatments is high and essentially unaffordable for Third World countries. The use of resistant varieties forms a useful alternative and should be developed further as a primary control option. Classical breeding has allowed the selection of several resistant varieties. However, for some crops, resistant varieties do not exist, while for most others resistance against only some species or pathotypes is found. Some of the commercially used resistance traits are temperature sensitive, and therefore unsuited for use in tropical and subtropical region@. More breeding efforts will be needed to achieve a satisfactory level of protection. On the other hand, the introduction of resistance traits from related wild species into commercial varieties by classical breeding is very time consuming. It is also usually difficult to separate the resistance trait from undesirable and agronomically unacceptable factors, linked to the resistance gene. The cloning of monogenie resistance genes and their transfer to other plant varieties or species will circumvent these problems and provide farmers with new resistant cultivars. It should also become possible, especially if information about the function of these genes is available, to engineer novel types of resistance (eg. with broader host ranges, or reduced temperature sensitivity) by molecular manipulations of the cloned resistance genes. It should be mentioned that monogenic resistance traits, with gene-for-gene relationships, are usually unstable under conditions of high selective pressurem. Other strategies, based on the study of plant genes induced during a compatible interaction, could lead to more stable resistances. The idea is to use promoters that are up-regulated in feeding cells to express proteins that would kill or harm the nematode, or the feeding cell on which the parasite depends. Currently, promoters expressed exclusively in feeding cells have not been identified and are not very likely to exist, for evolutionary reasons. To counterbalance the possible ‘leakiness’ of promoters induced in feeding cells but also expressed in different tissues or during specific phases of development, a two-component system has been designed61@. In such a system, the promoter of a gene expressed in nematode-induced feeding cells is fused to a ‘killer gene’ (such as an RNase). This construct is then transferred to plants, constitutively expressing a gene that would neutralize the effect of the ‘killer gene’ outside of the nematode feeding site. Preferably, the constitutive
Parasitology
Today,vol. IO, no. I I, I 994
promoter used should be down-regulated in feeding cells. Such down-regulation of several constitutive promoters has been observed inside syncytia29. A typical example of a two-component system, which has been used to engineer male-sterile plants, is the RNase barnase, coupled to its specific inhibitor barstafi3. Another possibility is to identify and isolate strictly nematode-responsive elements within nematode-upregulated promoters20 and to use these to drive the expression of a ‘killer gene’. The parasite’s point of -view Plant-parasitic nematodes have evolved morphological adaptations to parasitism, including a stylet (which allows them to pierce host tissues and to inject or withdraw fluids from plant cells) and highly specialized oesophageal glands (F$. 5). For both root-knot and cyst nematodes, a role for stylet secretions (saliva) in induction and maintenance of giant cells or syncytia has been suggested (for a review, see Ref. 64). Secretory granules from the dorsal gland, an organ that enlargens and becomes more active after the onset of the parasitic phase of the larva (Fig. 5), could play a crucial role65,M. Studies on nematode secretions have revealed the presence of at least nine proteins in stylet exudate of excised Meloidogyne adult female@. Monoclonal antibodies (mAbs) raised against isolated secretory granules from the dorsal gland of Meloidogyne have allowed the immunopurification of a 212 kDa glycoprotein. The exact function of this protein is still unknowr@. Monoclonal antibodies have been raised against specific structures of root-knot and cyst nematodes and larval stage-specific mAbs have been identifiedeg-71. A mAb specific for the amphids (an organ involved in chemoreception) of various Mebidogyne species” partly impairs the ability of nematodes to locate roots”. Besides their fundament,al interest for the understanding of the plant-nematode interactions, some mAbs could be used to engineer resistance via the ‘plantibody’ technology 74. The lidea is to express mammalian mAbs directed against precise antigens of the nematode within plants. Hiatt et al.75 first expressed whole antibodies in plants. Functional single-chain antibodies (ScAbs), which have fewer requirements for assembly, can be expressed to high levels in the apoplasm via the default secretion pathway76. This is encouraging for the engjneering of nematode resistance using mAbs, such as the amphid-specific mAbs, that could interfere with migrating larvae. Expression of antibodies in the cytosol leads to much lower ScAb accumulation, probably because of instability of the antibody76,n. However, this is still enough to cause delayed and reduced .virus infection symptoms78. Plants expressing mAbs directed against oesophageal gland secretions, which are possibly injected into the cytoplasm of future feeding cells, might therefore show some protection ag:ainst nematodes. Conclusions The interaction between plants and root-knot and / or cyst nematodes is studied intensively for both fundamental and applied reasons. The recent introduction of recombinant DNA and protein techniques into plant nematology, as well as the use of the model host system, A. fhakzna, has opened new perspectives. On the plant side, genes expressed in roots upon infection have
429 been isolated and a picture of the complex molecular changes induced by nematodes has started to emerge. In addition, resistance genes have been mapped and their cloning by chromosome walking has been initiated. On the nematode side, some of the components thought to be involved in the induction of feeding cells have been isolated and studied. The approaches discussed in this paper are only beginning to yield valuable data and much remains to be understood about this fascinating plant-parasite interaction. However, these approaches have already contributed to the evolution of plant nematology from a more descriptive towards a more analytical phase. Acknowledgements The authors thank Tom Gerats and Jonathan Clarke for critical reading, Mat-tine De Cock for help with the manuscript, and Vera Vetmaercke and Karel Spruyt for the figures. This work was supported by a grant from the Vlaams Actieprogramma Biotechnologie (No. 073). GG is a Senior Research Assistant ofthe National Fund for Scientific Research (Belgium). References 1 Sasser, J.N. and Freckman, D.W. (1987) in Vistas on Nematology: A Commemoration of the Twenty-Ffth Anniversary of the Society of Nemafologists (Veech, J.A. and Dickson, D.W., eds), pp 7-14, Society of Nematologists Inc. 2 Despommier, D.D. (1990) PurusitoIogy Today 6,193-196 3 Bird, A.F. (1961) 1. Biophys. Biochem. Cytol. 11,701-715 4 Endo, B.Y. (1971) in Plant Parasitic Nematodes (Vol. II) (Zuckerman, B.M., Mai, W.F. and Rohde, R.A., eds), pp 91-117, Academic Press 5 Jones, M.G.K. and Dropkin, V.H. (1975) Physiol. Plant Puthol. 5, 119-124 6 Pate, J.S. and Gunning, B.E.S. (1972) Annu. Rev. Plant Physiol. 23, 173-196 7 Jones, M.G.K. and Northcote, D.H. (1972) J. CeZZSci. 10,789-809 8 Huang, C.S. and Maggenti, A.R. (1969) Phytopathology 59, 447-455 9 Bird, A.F. (1973) Physiol. Plant PathoZ. 3,387-391 10 Jones, M.G.K. and Payne, H.L. (1978) 1. NematoZ. 10,70-84 11 Huang, C.S. (1985) in An Advanced Treatise on Meloidogyne, VoZ.I: Biology and Control (Sasser, J.N. and Carter, C.C., eds), pp 155-164, Department of Plant Pathology and US Agency for International Development 12 Gurr, S.J. et al. (1991) Mol. Gen. Genef. 226,361-366 13 Niebel, A. et al. (1993) Arch. Int. Physiol. Biochim. Biophys. 101, B18 14 Lamb, C.J. (1994) Cell 76,419-422 15 Chen Z., Silva, H. and KIessig, D.F. (1993) Science 262, 1883 -1886 16 Niebel, A. et al. (1993) PZant Cell 5,1697-1710 17 Carpita, N.C. and Gibeaut, D.M. (1993) Plant ].3,1-30 18 Ye, Z-H. and Vamer, J.E. (1991) Plant Cell 3,23-37 19 Esquerr&Tugay& M.T. et al. (1979) PZunf Physiol. 64,320-326 20 Opperman, C.H., Taylor, C.G. and Conkling, M.A. (1994) Science 263,221-223 21 Sijmons, P.C. et al. (1991) Plant ].1,245-254 22 Somerville, C. (1989) Plant CeZZ1,1131-1135 23 Ferreira, P.C.G: et aZ: (1991) Plant CeZZ3,531-540 24 Hemerlv. A.S. et al. (1993) Plant Cell 5.1711-1723 25 Niebel,‘i. et al. h A&nces in kIoZecuZar Plant Nemafology (NATO-AS1 Series) (Lamberti, F., De Giorgi, C. and McBird, D., eds), Plenum Press (in press) 26 Rubinstein, J.H. and Owens, R.G. (1964) Confrib. Boyce Thompson Inst. 22,491-502 27 Endo, B.Y. (1971) Phytopathology 61,395-399 28 Jasmer, D.P. (1993) J. Cell BioZ. 121,785-793 29 Goddijn, O.J.M. et al. (1993) Plant J. 4,863-873 30 Bird, A.F. and Loveys, B.R. (1975) 1. NematoZ. 7,111-113 31 Jones, M.G.K. et al. (1974) Protoplasma 80,401-405 32 Jones, M.G.K. (1981) Ann. AppZ. BioZ. 97,353-372 33 Lindsev, K. et al. (1993) Trunsaenic Res. 2,33-47 34 Meyer&vitz, E.M: (1989) CeZZ>, 263-269 35 Siimons. P.C.. von Mende. N. and Grundler. F.M.W. in Aiabiddpsis (Somenrille, C.’ and Meyerowitz, E., eds), Cold Spring Harbor Laboratory Press (in press)
PorositologyToday, vol. IO, no. I I, I994
430 36 Dangl, J.L. et al. (1992) in Methods in Arabidopsis Research (Koncz, C., Chua, N-H. and ScheII, J., eds), pp 393-418, World Scientific 37 Niebel, A. et al. (1993) in Mechanisms of Plant Defense Responses (Development in Plant PuthoZogy, Vol. 2) (Fritig, B. and Legrand, M., eds), pp __ 344-348, Kluwer Academic Publishers 38 Dolan, L. et al. (1993) Development 119,71-84 39 Wvss. U. and Gnmdler. F.M.W. (19921 Nematologica 38.488-493 40 W&s) U., Grundler, F.M.W. ani M&h, A. (1932) N&natologica 38,98-111 41 BBckenhoff, A. and Grundler, F.M.W. Parasitology (in press) 42 Liane. I?. and Pardee. A.B. (1992) Science 257,967-971 43 Sidh;, G.S. and Webster, J.&I. (1481) Bat. Rev. 47,387-419 44 Robinson, M.P., Atkinson, H.J. and Perry, R.N. (1988) Rev. NtfmatoI. 11,99-107 45 Kim, Y.H., Riggs, R.D. andKim, K.S. (1987)J. Netnatol. 19,177-187 46 Rice, S.L., Stone, A.R. and Leadbeater, B.S.C. (1987) Physiol. Mol. Plant Pathol. 31, l-14 47 Bleve-Zacheo, T., MeliIlo, M. and Zacheo, G. (1990) Rev. Nt+natol. 13, 29-36 48 Endo, B.Y. (1991) Rev. Ntfmutol. 14,73-94 49 Golinowksi, W. and Magnusson, C. (1991) Can. J. Bat. 69,53-62 50 Hammond-Kosack, K.E., Atkinson, H.J. and Bowles, D.J. (1990) Physiol. Mol. Plant PathoI. 37‘339-354 51 Barone, A. et al. (1990) Mol. Gen. Genet. 224,177-182 52 Pineda, O., Bonierdale, M.W. and Plaisted, R.L. (1993) Genome 36,152-156 53 Gebhardt, C. et al. (1993) Theor. Appl. Genet. 85,541-544 54 Messeguer, R. et al. (1991) Theor. Appl. Genet. 82,529-536 55 Ho, J-Y. et al. (1992) Plant J. 2,971-982
56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78
van Daelen, R.A.J.J. et al. (1993) Plant Mol. Biol. 23, 185-192 Jung, C. et al. (1992) Mol. Gen. Genet. 232,271-278 Salentijn, E.M.J. et al. (1992) MoI. Gen. Genet. 235,432~440 Roberts, P.A. (1992) 1. Nematol. 24,213-227 Jarquin-Barberena, H. et UI.(1991) Rev. N&nutoZ. 14,261-275 Sijmons, I’. et al. (1993) Patent Application No. PCT/EP92/02559, WO 93/ 10251 Van der Eycken, W. et al. (1992) Patent Coop. Treaty EP/92/01214, Int. Publ. No. WO 92 /21757 Mariani, C. et al. (1992) Nature 357,384-387 Hussey, R.S. (1989) Annu. Rev. Phytupathol. 27,123-141 Endo, B.Y. (1987) 1. Nematol. 19,469-483 Hussey, R.S. and Mims, C.W. (1990) ProtopIusmu 156,9-18 Veech, J.A., Starr, J.L. and Nordgren, R.M. (1987) 1. Nemutol. 19, 463-468 Hussey, R.S., Paguio, O.R. and Seabury, F. (1990) Phytopathology 80,709-714 Atkinson, H.J. and Harris, P.D. (1989) Parasitology 98,479-487 Davis, E.L. et uI. (1992) Phytoputhology 82,1244-1250 Backett, K.D., Atkinson, H.J. and Forrest, J.M.S. (1993) J. Nemutol. 25,395-400 Stewart, G.R., Perry, R.N. and Wright, D.J. (1993) Parasitology 107,573-578 Stewart, G.R. et al. (1993) Parasitology 106,405-412 Schots, A. et al. (1992) Neth. J. Plant Pathol. 98 (Suppl. 2), 183-191 Hiatt, A., Cafferkey, R. and Bowdish, K. (1989) Nature 342,76-78 Firek, S. et al. (1993) Plant Mol. Biol. 23,861-870 Owen, M. et al. (1992) BiolTechno2ogy 10,790-794 Tavladoraki, I’. et al. (1993) Nature 366,469-472
The Role of Salivary Vasodilators in Bloodfeeding and Parasite Transmission D.E. Champagne In this paper, Donald Champagne reviews the salivary vasodilators, points to effects of similar compounds that may be shared by the insect substances, and discusses the potential significance of these effects with regard to parasite transmission. Bloodfeeding is a complex task made difficult by the presence of hemostatic mechanisms used by vertebrate hosts to prevent blood 1ossQ. In the case of small lesions, such as those produced by a feeding arthropod, the most significant components of hemostasis are: (1) the formation of a platelet plug (in response to ADP and collagen); and (2) vasoconstriction. Although the hematophagous habit has arisen independently many times, all arthropods that have been examined to date use the enzyme apyrase to inhibit platelet aggregation by hydrolysing ADP and ATPQ. In contrast, a remarkable diversity of substances have been employed as vasodilators (for summary see Table 1). In several instances, these substances are close mimics of, or identical to, endogenously produced signal molecules that affect systems other than vasodilation, including components of the immune response.
Donald Champagne is at the Department of Entomology, University of Arizona, Tucson, AZ 8572 I, USA.
Mosquitoes The salivary vasodilator of the yellow fever vector Aedes aegypti, first noted by Pappas et a1.3, was subsequently characterized as a tachykinin based on its endothelium dependence, cross-desensitization with the mammalian tachykinin substance P and reaction with an anti-substance I.’antibodyd. When the vasodilator was purified and sequenced, it was shown to comprise two novel tachykinins, sialokinin I and II, the first tachykinins to be isolated from an insects. Structurally, these peptides combine characteristics of substance P with those typical of neurokinins, the second major subgroup of mammalian tachykinins. These peptides were as potent as substance P in assays on the guinea-pig ileum. The Lacrosse Fever vector, Aedes triseriatus, also employs a tachykinin, which differs from the sialokinins in its chromatographic properties6. Indeed, the use of vasodilatory tachykinins may be characteristic of the genus Aedes. Substance P is the most abundant tachykinin in skin, where its primary role is as a sensory neuromodulator. In addition, substance P and other tachykinins including the neurokinins stimulate the oxidative burst and thromboxane release from macrophages7JJ. Of particular interest, both neurokinin A and (to a lesser extent) substance P enhance the primary antibody response in macrophages: maximum effect was seen at 10~~ but responses were produced by 0
1994, Elsev,er Sc,ence Ltd