Rice Genetics Newsletter 12 (1995) 155
E. Report of the Committee on Genetic Engineering (Molecular Analysis of Rice Genes)
Ray Wu.
Convener
Section of Biochemistry,. Molecular & Cell Biology. Cornell University.
lthaca. NY 14853. U.S.A.
     This year's genetic engineering report briefly reviews the progress made in cloning and molecular characterization of disease-resistant genes (R genes) in plants. The report is divided into four sections. The First section described the importance of cloning R genes: the second section briefly outlines the available methods for cloning R genes from plants: the third section gives several examples of cloning and molecular characterization of several R genes: and the last section is a brief summary, including future prospects.

I. The Importance of Disease-Resistant Genes (R Genes) in Plants

      Resistance of a plant to a pathogen is often correlated with a hypersensitive response, such as localized, induced cell death in the host plant at the site of infection. In gene-for-gene interactions between a plant and a pathogen. resistance is expressed only when a plant containing a specific R gene recognizes a pathogen that has the corresponding avirulence gene (for a review, see Staskawicz et al. 1995). Physiological features of hypersensitivity include a rapid oxidative burst. K+-H+ exchange, crosslinking of plant cell wall. synthesis of antimicrobial compounds such as phytoalexins. and induction of pathogenesis-related proteins such as chitinases and glucanases (Lamb et al. 1989). The mechanism by which these events limits the growth of specific pathogens has not yet been elucidated.

       Avirulence genes corresponding to specific R genes were cloned from bacterial and fungal pathogens ten years ago (Staskawicz et al. 1984: Gabriel et al. 1986). However. since little is known about the protein products of R genes in plants, the isolation and cloning of R genes remains difficult. In fact. the first R gene in plants was not cloned until 1992 (Johal and Briggs 1992).

      Cloning plant R genes opens the door for producing disease-resistant crop plants in two ways. First, by using cloned R genes as probes, plant breeders can monitor R gene segregation more readily for disease resistance and susceptibility. Moreover, the cloned R genes can be used to facilitate the identification and introgression of new resistances from wild plant species. Second, using direct genetic engineering of crop plants by transforming specific R gene(s) into plants, one can produce disease-resistant plants with relative ease.

II. Methods for Cloning Disease-Resistant Genes (R Genes) from Plants

There are two general ways to clone an R gene. or any gene. when no information is available about the gene product: insertional mutagenesis and map-based cloning.

A. Insertional mutagenesis—In this method, a fragment of DNA is inserted into the coding region or regulatory region of a gene.
        which results in the disruption of the gene expression. After the insertional mutagenesis. the next step is to clone the plant DNA
        that flanks the integrated insertional mutagen by plasmid rescue. The cloned plant DNA can be used as a hybridization probe to
        isolate the gene by screening a lambda or cosmid library constructed from the wild-type plant. If the gene of interest is a
        disease-resistant gene, the final test for the gene tagged by this method is to introduce the cloned gene from the disease-resistant
        plant by transforming sensitive plants and examining them in order to determine whether they have become disease-resistant.
1. The most commonly used DNA molecules for insertional mutagenesis are transposons. Fortunately, members of the maize Ac and
        Spm transposon families function when transferred into heterologous plant species. This attribute allows for efficient gene-tagging
        systems in a variety of plants specific for tagging and cloning genes of interest (Baker er al. 1986).
2. Another commonly used insertion mutagen is the T-DNA from the Ti plasmid. After infection of plant cells by Agrobacterium,
        T-DNA plus any gene (such as the NPTII gene) inserted between the 25-base pair borders that flank the T-DNA become
        integrated into the plant genome. After selection with kanamycin. a certain percentage of the resistant plant lines exhibited a
        mutant phenotype as the result of gene disruption.
B. Map-based cloning—In map-based cloning (also known as positional cloning), one needs to first determine the approximate
        chromosomal location of a gene of interest. The most frequently used method for linking the chromosomal location to a particular
        trait, such as resistance to a specific disease in a plant, is to make use of restriction fragment length polymorphism (RFLP).
        Co-inheritance of the disease resistance trait with one or several specific RFLP DNA probes defines the approximate location of
        the R gene on a specific chromosomal region. The linkage between several specific DNA probes and the R gene allows one to
        use the DNA probes as starting points to walk to. or to land on. the R gene. The R gene is provisionally identified after screening
        for coding sequences (e.g.. cDNAs) within the specific region, preferably cloned in a yeast artificial chromosome (YAC) vector
        or a bacterial artificial chromosome (BAC) vector. Finally, verification of the putative R gene can be determined by genetic
        complementation tests after transforming the candidate cDNA fragments into susceptible plants and after looking for the
        acquisition of disease resistance in the transgenic plants. Alternatively, the R gene can be verified by isolating mutants in the R
        gene and comparing the mutant sequences with the wild-type sequence,

III. Molecular Characterization of Several Disease-Resistant (R) Genes

A. Identification of R genes by insertional mutagenesis—

1. The first plant R gene to be cloned by insertional mutagenesis was the maize Hml

(Johal and Briggs 1992). This gene, which controls resistance to the fungus Cochliobolus carbonum race 1. was identified by transposon tagging with the Mu transposon of maize. After cloning, Hml was found to encode a NADPH-dependent HC toxin reductase (HCTR). which inactivates the HC toxin produced by the fungus. Several Hml alleles were generated and cloned by transposon-induced mutagenesis. The sequence of the wild-type Hml shares identity with dihydroflavonol-4-reductase genes from maize, petunia, and snapdragon. 2. Three additional plant R genes were cloned using the insertional mutagenesis approach. The tobacco N gene, which confers
            resistance to the viral pathogen tobacco mosaic virus (TMV), was isolated by transposon tagging with Ac (Whithman et al.
            1994), A genomic DNA fragment conatining the N gene conferred TMV resistance to TMV-susceptible tobacco. Sequence
            analysis of the N gene shows that it encodes a 131-kDa protein with an amino-terminal domain similar to the interleukin-1
            receptor in mammals: a nucleotide-binding site. and four imperfect leucine-rich repeats.
3. The tomato Cf-9. which confers resistance to the fungal pathogen C.fulvum expressing the avirulence gene avr9, was tagged by a
            maize Ds transposable element (Jones et al. 1994). A tomato line lacking Cf-9 was engineered that expressed the C.fulvum
            avr9 gene under the control of a plant gene promoter. When this line was crossed with a line containing both Cf-9 and a Ds
            element, most of the progeny died because the interaction of the avr gene product with the Cf-9 gene product resulted in the
            elicitation of a systemic hypersensitive response. However, mutants carrying a Ds-inactivated tagged Cf-9 gene survived.
4. The flax L6 gene, which confers resistance to the fungal pathogen M. lini, was identified by tagging with the maize transposon Ac.
            Mutants were identified by visual inspection of thousands of flax plants containing putative transpositions
            of Ac into the L6 gene (Ellis et al. 1995).
B. Identification of R genes by map-based cloning—
1. In plants, the first R gene to be cloned by the map-based cloning method was the tomato PTO (Martin et al. 1993). The PTO in
            tomato confers resistance to races of Pseudomonas syringae pv. tomato that carry the avirulence gene avrPto.
            A 400-kb YAC clone (PTY538-1). which spans the Pto region, was identified by an RFLP marker. TG538.
            that cosegregated with the Pto locus. A sample of DNA from PTY538-1 was used to probe a leaf cDNA
            library, and 30 hybridizing plaques were investigated further. The cDNA inserts in these plaques were used to
            probe a tomato mapping population consisting of 85 plants with recombination events in the Pto regions. When
            the clone CD    127 was mapped, it cosegregated with Pto. Finally, genetic complementation tests were carried
            out to determine if CD 127 confers resistance to P. syringae pv. tomato. After transforming a susceptible tomato
            cultivar. plants were inoculated with P. syringae pv. tomato strains carrying avrPto. Of two plants that were
            confirmed to contain the integrated transgene, both were resistant to this pathogen. The coding region of this R
            gene was sequenced, and the deduced amino acid sequence encodes a 321-amino acid hydrophilic protein. The
            protein sequence showed similarity to serine-threonine protein kinases. suggesting a role for Pto in a
            signal-transduction pathway.
2. An additional plant R gene was cloned. The Arabidopsis RPS2 gene. which confers resistance to the bacterial
            pathogen P. syringae pvs. tomato and maculicola expressing the avirulence gene avrRpt2. was identified by
            isolating Arabidopsis mutants that did not exhibit a hypersensitive response after infection by P. syringae
            strains carrying avrRpt2. The RPS2 gene was then cloned using the map-based strategy (Bent et al. 1994:
            Mindrinos et al. 1994).
3. The Arabidopsis RPMI gene was cloned by positional cloning after RFLP mapping. followed by transformation
            and by looking for complementation (Grant et al. 1995). The RPMI gene was identified within the minimum
            complementing region by sequencing genomic DNA from Col-0 DNA and four mutant rpml alleles. In 4.5 kb
            of wild-type DNA. one large open reading frame (ORF) of 2778 bp was identified. The RPMI ORF contains
            features found in the predicted polypeptide sequences of other R genes: a potential leucine zipper, two motifs
            of a nucleotide-binding site. and 14 imperfect leucine-rich repeats. These features most closely resemble those
            of the A. thaliana RPS2 gene. which confers resistance to P. syringae expressing avrRpt2. The RPMI gene
            enables dual specificity to pathogens expressing either of two unrelated P. syringae avr genes.
4. The first rice R gene to be cloned by the map-based cloning method was the Xa21 gene, which confers resistance
            to Xanthomonas oryzae pv. oryzae race 6. Fifty transgenic rice plants carrying the cloned Xa-21 gene display
            high levels of resistance to the pathogen (Song et al. 1995). The sequence of the predicted protein, which
            carries both a serine-threonine kinase-like domain and a leucine-rich repeat motif, strongly suggests that it plays
            a role in cell surface recognition of a pathogen ligand and subsequent activation of an intracellular defense
            response. Characterization of the Xa-21 gene will facilitate our understanding of plant disease-resistance and lead
            to genetically engineered resistance in rice.
    It may be of interest to note that two primitive procedures were proposed in 1986 for cloning disease resistance genes through the transgenic rice approach, with and without  prior RFLP analysis (Wu et al. 1987). At that time. the YAC library technology was not yet fully developed, and the regeneration of rice plants was difficult. Over the last nine years, a high-density RFLP map for rice has become available, together with rapid advancements in molecular biology and transformation technology, cloning of disease resistance genes by improved map-based cloning procedures is now a reality.
IV. Summary and Future Prospects
            In this brief review, it is clear that disease-resistant genes can be cloned from higher plants. Within the last three years, spectacular progress has been made in the molecular cloning of eight disease-resistant genes. Yet. the task of first identifying and then cloning an R gene is rather labor-intensive and time consuming. It often takes 5 to 7 skilled scientists 2 to 3 years to clone an R gene. However, one can expect that many more R genes will be cloned before the end of this century because knowing their structure will lead to a better understanding of pathogen-host interactions. Moreover, the availability of cloned R genes will increase the potential for engineering disease resistance in plants.

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