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Proceedings of a Workshop on: 

Ecological Effects of Pest Resistance Genes in Managed Ecosystems

January 31 - February 3, 1999 Bethesda, Maryland

Organized by: Information Systems for Biotechnology

MECHANISMS OF PEST RESISTANCE IN PLANTS 1

Noel Keen

University of California-Riverside

INTRODUCTION

In their long association with pests and pathogens, plants evolved an impressive array of defensive tools. At the same time, pests and pathogens developed mechanisms to compromise plant resistance mechanisms in what must have been an evolutionary game of ping-pong. Natural pest resistance mechanisms occurring in higher plants can be classified into preformed resistance mechanisms and inducible resistance mechanisms. Agricultural pest control throughout this century has attempted to harness these mechanisms wherever possible. Natural resistance has several obvious advantages over the use of chemical pesticides or other methods for pest control. These include nominal genetic permanency, negligible cost once cultivars are developed, and quite high efficacy. The major downside of natural pest resistance is the reality that selection pressure is placed on pest populations to develop means of overcoming the resistance, thus practically limiting the time of effectiveness.

PREFORMED RESISTANCE MECHANISMS

Resistance mechanisms of this type are usually broken down into preformed structural, morphologic, and chemical factors. In entomology, it has long been known that innate morphological and anatomical features such as leaf and flower color, presence of trichomes, and even the texture of cuticle may cause certain insects to avoid a plant, thus constituting resistance mechanisms. Anatomical features may also deter or discourage insect feeding. These include the degree of secondary wall thickening, stelar structure, and other aspects of basic plant structure. They all fall under the category of preformed resistance mechanisms.

Plant pathogens include viruses, fungi, bacteria, and nematodes, all of which must gain entry into the plant and contact living plant cells in some way for success. Accordingly, structural and morphological barriers could be expected to provide resistance against many potential invaders. Recognized examples include features as sophisticated as stomatal guard cell anatomy, for instance the height of lips of the guard cells. As shown in work by Harvey Hoch and colleagues (Hoch et al. 1987) at Cornell University, certain fungal rust pathogens initially colonize the surface of leaves and have exquisite sensing mechanisms that measure the height of stomatal guard cell lips encountered on susceptible plants. When the fungus hyphae encounter a lip of the proper height, they are programmed to undergo a developmental program resulting in the formation of invasive structures that enter the stomate and begin colonization of the leaf interior. It has been noted that if one could alter the height of guard cell lips, this rather benign change should provide resistance against the rust fungus.

Plants typically contain significant amounts of preformed chemicals produced via secondary metabolism. These include phenolics of varying structural sophistication, terpenoids, and steroids. The concentrations of these compounds in particular tissues may be very high. Some preformed compounds are directly toxic, while others exist as conjugates such as glycosides that are not directly toxic but become toxic following disruption of the conjugate. For instance, plant glycosides are often hydrolyzed following insect damage or pathogen ingress that releases vacuolar glycosidases. The aglycones thus produced may be quite toxic to the invader as well as neighboring plant cells. Since the toxic response is local, however, only a small portion of the plant is affected.

On the other hand, some plant preformed compounds are toxic as glycosides, but lose toxicity when deglycosylated. Elegant work done with fungal plant pathogens has proven the role of several such compounds as bona fide resistance factors. In one example, the preformed saponin glycoside, avenacin, was shown to inhibit the growth of a root pathogenic fungus, and oat plants producing the compound exhibited resistance to the pathogen. A related fungus strain, however, was observed to produce a glycosidase that removed the sugar residue from avenacin, effectively detoxifying it. This strain was not inhibited by avenacin and oat plants were susceptible to it. Anne Osborne and colleagues (Bowyer et al. 1995) at the John Innes Institute in England cloned the gene for the fungal glycosidase from the detoxifying strain and showed that mutation of this gene rendered the fungus sensitive to growth inhibition by avenacin. More importantly, oat plants were now resistant to the mutant strain, strongly arguing that avenacin is a resistance factor unless a pathogen can deal with it.

INDUCIBLE RESISTANCE MECHANISMS

Inducible resistance mechanisms are active, energy-requiring systems typified by specific recognition of an invader that ultimately leads to the production of proteins or metabolites that are antagonistic to the invader. These resistance mechanisms have been most studied in regard to plant pathogens, but the same or similar mechanisms clearly function against insect pests. Such active resistance mechanisms are usually referred to collectively as the hypersensitive response (HR).

RECOGNITION ASPECTS IN THE HR

Invocation of the HR requires that the plant recognize or key on at least one molecule produced by the invading pest. These factors have come to be called elicitors and may be peptides or proteins, fatty acid derivatives, sterols, or other low molecular weight chemicals produced by a pest or pathogen. Elicitors themselves, in the absence of the living pests, initiate the active plant defense response.

Plants have been known since early in this century to contain particular genes, called disease resistance genes, that confer resistance to some but not all biotypes or strains of a pest or pathogen. These genes have been widely used in practical agriculture, and have allowed farmers to avoid using countless tons of chemical pesticides. There are, unfortunately, cases where certain plants do not have an identified resistance gene against an important pest, and pesticides still have to be used. There is also the problem of pests mutating to virulent forms that are no longer recognized by the disease resistance gene, effectively rendering it useless. Strains of pathogens that initiate plant defenses harbor genes called avirulence genes. These genes direct the production of specific elicitors, which when purified, have the rather remarkable property of initiating the HR only in plant cultivars containing the cognate or matching disease resistance gene. Pest strains that have escaped resistance conferred by a certain plant resistance gene have either eliminated production of an elicitor by losing the corresponding avirulence gene or (if the elicitor is a protein) have modified its structure such that the resistant plant no longer detects it.

In the last few years, many different plant disease resistance genes have been cloned and sequenced. Almost all of them fall into the leucine-rich repeat (LRR) class of proteins, typified by imperfect repeats of blocks of amino acids, usually with about 24 residues per repeat element. The LRR resistance gene proteins may also have nucleotide binding sites, leucine zipper domains, or kinase domains suggestive of signal transduction functions. In a few cases, disease resistance genes have been transferred to foreign plants by transformation and generally shown to be functional. Although no commercial plant cultivars have yet been developed, it is suspected that transfer of disease resistance genes by transformation will become a commonly used method to develop new pest-resistant plants.

A few LRR plant disease resistance genes have been shown to exhibit dual specificities—that is, the plant harboring them either recognizes two different pests or two different elicitors. Especially exciting was the recent finding by Valerie Williamson and colleagues at the Univ. of California, Davis (Rossi et al.1998) that the cloned Mi resistance gene in tomato against the root knot nematode also recognizes a species of aphid. It is not known whether the nematode and aphid produce the same elicitor, as is likely, but the finding is of considerable importance and has practical implications that should stimulate the search for additional disease resistance genes that target insects. While several examples of insect-targeting resistance genes are recognized, they are relatively rare compared to resistance genes known against fungi, bacteria, nematodes, and viruses.

INDUCED RESISTANCE RESPONSES

When resistant plants recognize cognate or matching elicitors, intracellular signal transduction pathways are activated that ultimately result in the derepression of a battery of genes called defense response genes. These latter genes encode toxic proteins such as chitinases, glucanases, lysozyme-active proteins, or cell wall strengthening proteins such as hydroxyproline rich glycoproteins. Response proteins may also be enzymes in biosynthetic pathways for lignification of cell walls or the production of phytoalexins, low molecular weight toxic chemicals that antagonize the invader.

Our knowledge of signal transduction in the HR is incomplete, but several interesting genes have been identified from mutagenesis screens or biochemical studies. These genes include protein kinases and phosphatases, calmodulin genes, and others of unknown biochemical function that ultimately activate transcriptional activators of defense response genes.

FUTURE APPLICATIONS

The Arabidopsis genome sequencing project should be completed by the end of 1999. These results will add significantly to the repertoire of genes available for producing transgenic plants. Indeed, understanding the functions of unknown genes identified by the sequencing project will be greatly aided by routinely transforming them into the same or heterologous plants and screening the resulting transgenics for various traits, including pest and pathogen resistance. We can accordingly expect a revolution in approaches to improvement of plants by the construction of transgenics.

There are several strategies that are being evaluated for harnessing what is known about active pest and disease resistance to crop improvement. There have been attempts by conventional plant breeding to introduce genes that alter the morphologic or chemical composition of plants such that they become unattractive to pests and pathogens. I suspect that much more of this kind of work will occur now that it is possible to routinely produce transgenic plants and, in principle, introduce genes for new biochemical pathways.

Several investigators have transformed pathogen avirulence genes responsible for production of elicitors into plants that carry the cognate disease resistance gene. There are some clever approaches underway in this arena, generally involving wound or defense response gene promoters used to regulate expression of the avirulence genes, such that they will only be expressed (and the HR elicited) following pathogen challenge. Various viral genes, such as coat or replicase genes, have also shown promise for producing resistance when transformed into plants.

Several HR signal transduction genes have been experimentally over-expressed in transgenic plants and some of them lead to enhanced pest and disease resistance. Accordingly, these genes are being studied for possible use in future disease resistant plant cultivars.

The LRR domains of disease resistance gene products have been shown to account for the specificity of these proteins to recognize only one pest elicitor. Thought has consequently been given to designing synthetic resistance genes with the LRR domains targeted to a certain elicitor of a pest or pathogen. Although this approach has not yet progressed beyond the experimental stage, it is clearly an area that will be heavily investigated in the future to generate new and unique disease resistance genes, hopefully some of them targeted to currently refractory pests and pathogens.

ECOLOGICAL CONCERNS

There is naturally the concern that heterologous natural disease resistance genes, engineered resistance genes, or synthetic resistance genes could be passed to weed populations and accordingly present hazards. Several factors make me think such dangers are minimal. First, natural disease resistance genes have been used throughout this century for pest and pathogen control and I know of no case where horizontal transfer of these genes has led to new weed problems. Intrinsically, there is no reason to think that engineered or synthetic resistance genes should behave any differently. Secondly, the big tactical advantage of creating transgenic plants is that genes of interest can usually be introduced into elite cultivars directly and relatively rapidly. Unlike classical plant breeding, this process drastically reduces the requirement for backcrosses and testing before a new cultivar can be released. The result of this ‘time-line shortening’ will be the ability to rapidly change the resistance genes present in crop plants, thus confounding pests and pathogens and their efforts to evolve and overcome resistance. This sleight of hand will also minimize the dangers of horizontal gene transfer. Newly inserted genes can be removed rapidly by simply substituting transgenics with new resistance genes for the old cultivars. As such, exposure time of any one gene can, in theory, be minimized, and pathogens accordingly will have less time to overcome it.

References:

Bowyer P, Clarke BR, Lunness P, Daniels MJ, and Osbourn AE. 1995. Host range of a plant pathogenic fungus determined by a saponin detoxifying enzyme. Science 267:371-374.

Hoch H, Staples RC, Whitehead B, Comeau J, and Wolf ED. 1987. Signaling for growth orientation and cell differentiation by surface topography in Uromyces. Science 235:1659-1662.

Rossi M, Goggin FL, Milligan SB, Kaloshian I, Ullman DE, and Williamson VM. 1998. The nematode resistance gene Mi of tomato confers resistance against the potato aphid. Proceedings of the National Academy of Sciences, USA 95:9750-9754.

General Reference on Active Disease Resistance in Plants:

Baker B, Zambryski P, Staskawicz B, and Dinesh-Kumar SP. 1997. Signaling in plant-microbe interactions. Science 276:726-733.

References on Transgenic Plants to Improve Disease and Pest Resistance:

Dixon RA, Lamb CJ, Masoud S, Sewalt VJH, and Paiva NL. 1996. Metabolic engineering: prospects for crop improvement through the genetic manipulation of phenylpropanoid biosynthesis and defense responses–a review. Gene 179:61-71.

Mourgues F, Brisset M-N, and Chevreau E. 1998. Strategies to improve plant resistance to bacterial diseases through genetic engineering. Trends in Biotechnology 16:203-210.

Schuler TH, Poppy GM, Kerry BR, and Denholm I. 1998. Insect-resistant transgenic plants. Trends in Biotechnology 1:168-175.

1 Paper presented at the "Workshop on Ecological Effects of Pest Resistance Genes in Managed Ecosystems," in Bethesda, MD, January 31 - February 3, 1999. Sponsored by Information Systems for Biotechnology.

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