P~ APT
C. EXPRESSION OF SPECIFIC HOST RESPONSE PROTEINS
A large number of proteins has been demonstrated to accumulate in barley leaves attacked by Bgh, either immunologically, by increase in enzyme activity, or indirectly in gene transcript analyses (see Collinge et ai. 1997). These include the pathogenesis-related (PR) proteins: PR-l's (Muradov et aI., 1993; Bryngelsson et aI., 1994), f3-1,3-glucanases (Jutidamrongpham et aI., 1991), chitinases (Kragh et aI., 1990, 1993), PR-4's (Gregersen et aI., 1997) and PR-5's (Bryngelsson and Green, 1989; Gregersen et aI., 1997). Members of the antimicrobial thionins (Bohlmann et aI., 1988) and lipid transfer proteins (Molina and Garcia-Olmedo, 1993) have also been found to accumulate in these leaves. So too have enzymes involved in secondary metabolism, such as PAL (Shiraishi et ai., 1989; Clark et aI., 1994; Boyd et aI., 1994a, 1994b, 1995), chalcone synthase (CHS) (Gregersen et ai., 1997; Christensen et aI., 1998b), flavonoid and caffeic acid O-methyltransferases (OMT) (Gregersen et aI., 1994; Christensen et aI., 1998a) and peroxidases (Kerby and Somerville, 1989, 1992; Thordal-Christensen et ai., 1992; Scott-Craig et ai., 1995). Other proteins potentially involved in regulation of the defense response also accumulate, e.g. the 14-3-3 protein (Brandt et al., 1992), oxalate oxidases (Zhang et aI., 1995; Dumas et al., 1995;
Zhou et aI., 1998) and an oxalate oxidase-like protein (Wei et aI., 1998). Oxalate oxidases, and possibly also the oxalate oxidase-like protein, a{e generators of HP2. ~02 is a potential activator of gene expression and HR (see Mehdy, 1994). The accumulation of a chaperone located in the endoplasmic reticulum, the GRP94, is suggested to be related to the dramatic increase in protein export during the manifestation of defense response (Walther-Larsen et aI., 1993).
Studies of the gene transcripts for these proteins in barley leaves being attacked by Bgh have revealed that each gene transcript has its own unique accumulation pattern.
However, many common characteristics exist among the accumulation patterns.
For almost all, there are peaks at the time of papilla formation in response to the PGT and the appressorium, i.e. 4-8 hours and 12-24 hours, respectively (Davidson et aI., 1988; Brandt et aI., 1992; Thordal-Christensen et al.; 1992; Walther-Larsen et aI., 1993;
Clark et al., 1993, 1994, 1995; Boyd et aI., 1994a, 1994b, 1995; Scott-Craig. et aI., 1995; Gregersen et aI., 1997; Zhou et aI., 1998). The 12-24-hour peak is possibly, in part, HR-correlated, but it is very signifi~ant in susceptible plants also, which suggests the relation of this peak to papilla formation subjacent to the appressorium. By comparing the expression in susceptible and HR-resistant leaves, it has been documented for a
92 H. Thordal-Christensen, P.L. Gregersen & D.H. Collinge
number of the gene transcripts that they also have HR-correlated expression. This is so for the gene transcripts encoding PR-l, j3-1,3-glucanases, chitinase, PR-5, pe oxidase, PAL and thionin (Davidson et aI., 1988; Clark et aI., 1993; Boyd et ai., 1994a, 1994b, 1995).
These HR-correlated expressions appear 1 to 3 days after inoculation followed by a decline. The timing of this expression varies somewhat among laboratories, but seems related to the type of the HR (single or multi-cell). Similar HR-correlated gene transcript expression has been suggested for a number of the remaining response proteins mentioned above (Gregersen et ai., 1997). In addition, much of the work demonstrates that these gene transcripts, which show HR-correlated expression, are also expressed during early colony development on susceptible leaves, i.e. 3 to 5 days after inoculation.
In summary, most of the described gene transcripts encoding defense response protein show three distinct waves in their expression, one in response to the PGT, one in response to the appressorium and one, which is either HR-correlated in the resistant plants or appearing in response to colony formation in susceptible plants. Certain of the gene transcripts encoding defense response protein are clear exceptions from this three-wave pattern. A gene transcript has been identified which is exclusively papilla- correlated, i.e. a gene transcript encoding an oxalate oxidase-like protein (Gregersen et aI., 1997; Wei et aI., 1998) while 14-3-3 gene transcripts show a similar characteristic (Brandt et aI., 1992). In contrast, four gene transcripts are expressed either at the time of HR in resistant leaves or at the time of colony development in susceptible leaves, i.e. the gene transcripts encoding flavonoid OMT, CHS, PR-l and basic PR-5 (Gregersen et aI., 1997).
In relation to the powdery mildew fungus, which is restricted to the leaf surface and the epidermal tissue, it is relevant to examine the location of the individual defense response components. In a study of separated epidermal and mesophyll tissues, a surprisingly low gene transcript level was observed in the epidermis for a number of the gene transcripts mentioned above (Gregersen et aI., 1997). This was primarily the case for gene transcripts encoding PR-protein. These data suggest that many accumulating gene transcripts represent a general response, which is not necessarily important for the defense towards this particular pathogen. This view is supported by the fact that many of these are highly expressed in compatible interactions at the time of colony development.
V. Conclusions
Much is now known about the interaction between barley and the powdery mildew fungus. This knowledge is largely descriptive: analyses of the timing of fungal development in relation to the timing of visible host responses, analyses of the genes in host and pathogen determining specificity, identification of gene transcripts accumulating during the defense response which encode antimicrobial proteins, and analyses of the chemical nature of components which accumulate in the key defense structures, the papillae and the HR cells. There is also an increasing body of evidence, mainly from the use of inhibitors of gene transcription, translation, specific enzymes and of cytoskeleton formation which confirm the importance of certain of these responses.
We know very little of the physiology of the fungus. We do not understand how the fungus persuades the host to allow it to establish the biotrophic relationship. We do not know how resistance works i.e. whether the possession (and timely activation) of a single component prevents the successful establishment of the parasite or whether resistance is a threshold event by which the combined action of a number of components is necessary to ensure resistance. Essentially nothing is known concerning the signal transduction pathways leading to activation of defense mechanisms. Nevertheless, the recent cloning of Ml-o (Biischges et ai., 1997) and identification of molecular markers linked to specific resistance genes, and other genes required for resistance, makes us expect substantial progress in this area within the next few years. An understanding of these biological processes will provide new inspiration for the development of new strategies for controlling the disease as well as giving an insight into a complex biological relationship.
It is expected that future studies using molecular techniques will focus on the identification of factors necessary for the establishment of different key phases of development of the fungus, for example, appressorium formation and initiation of the haustorium. Firstly, an understanding of these processes can be expected to lead to the identification of new targets for control of th~ disease. Secondly, remarkably little is understood of the biological processes underlying the establishment of abiotrophic relationship. Plant pathogens and symbionts (e.g. Giberella and Rhizobium) have taught us about plant growth and development (about the hormone giberellin and about nod factors as stimulants of organogenesis). As biotrophy depends on regulation and exploitation of the physiological processes taking place in the leaf, it is to be expected that, by understanding how the fungus regulates these processes to its own benefit, we will learn more about plants' own mechanisms for regulating their physiology.
Acknowledgements
We are grateful to our colleagues Drs. Fasong Zhou and Eigil de Neergaard for providing the electron micrograph, and to Professor Sigrun Hippe-Sanwald, Christian Albrechts University, Kiel, Germany, for discussion.
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