▪ Abstract In “gene-for-gene” interactions between plants and their pathogens, incompatibility (no disease) requires a dominant or semidominant resistance (R) gene in the plant, and a corresponding avirulence (Avr) gene in the pathogen. Many plant/pathogen interactions are of this type. R genes are presumed to (a) enable plants to detect Avr-gene-specified pathogen molecules, (b) initiate signal transduction to activate defenses, and (c) have the capacity to evolve new R gene specificities rapidly. Isolation of R genes has revealed four main classes of R gene sequences whose products appear to activate a similar range of defense mechanisms. Discovery of the structure of R genes and R gene loci provides insight into R gene function and evolution, and should lead to novel strategies for disease control.

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Plant Disease Resistance Genes

■ Abstract Many disease resistance (R) proteins of plants detect the presence of disease-causing bacteria, viruses, or fungi by recognizing specific pathogen effector molecules that are produced during the infection process. Effectors are often pathogen proteins that probably evolved to subvert various host processes for promotion of the pathogen life cycle. Five classes of effector-specific R proteins are known, and their sequences suggest roles in both effector recognition and signal transduction. Although some R proteins may act as primary receptors of pathogen effector proteins, most appear to play indirect roles in this process. The functions of various R proteins require phosphorylation, protein degradation, or specific localization within the host cell. Some signaling components are shared by many R gene pathways whereas others appear to be pathway specific. New technologies arising from the genomics and proteomics revolution will greatly expand our ability to investigate the role of R proteins in plant disease resistance.

Understanding the functions of plant disease resistance proteins.

The Mi locus of tomato confers resistance to root knot nematodes. Tomato DNA spanning the locus was isolated as bacterial artificial chromosome clones, and 52 kb of contiguous DNA was sequenced. Three open reading frames were identified with similarity to cloned plant disease resistance genes. Two of them, Mi-1.1 and Mi-1.2, appear to be intact genes; the third is a pseudogene. A 4-kb mRNA hybridizing with these genes is present in tomato roots. Complementation studies using cloned copies of Mi-1.1 and Mi-1.2 indicated that Mi-1.2, but not Mi-1.1, is sufficient to confer resistance to a susceptible tomato line with the progeny of transformants segregating for resistance. The cloned gene most similar to Mi-1.2 is Prf, a tomato gene required for resistance to Pseudomonas syringae. Prf and Mi-1.2 share several structural motifs, including a nucleotide binding site and a leucine-rich repeat region, that are characteristic of a family of plant proteins, including several that are required for resistance against viruses, bacteria, fungi, and now, nematodes.

The root knot nematode resistance gene Mi from tomato is a member of the leucine zipper, nucleotide binding, leucine-rich repeat family of plant genes.

More than 30 genes have been characterized from different plant species that provide resistance to a variety of different pathogen and pest species. The structures of most are consistent with a role in pathogen recognition and defense response signaling. Resistance genes are very abundant in plant genomes and most belong to tightly linked gene families. Evolution of R genes is driven by selection on allelic variation created by mutation and re-assorted by recombination between alleles and sometimes between different gene family members. Selection favors genes that can recognize pathogen avr gene products that are present in pathogen populations. Selection at linked gene families favors haplotypes with useful combinations of genes but a limited physiological cost to the plant. Future utilization of R genes will include transfer between related genera and identification or construction of genes that condition durable resistance to variable pathogens. Genes with durable resistance may interact with conserved pathogen elicitors or condition resistance responses that are independent of specific Avr gene interactions.

RESISTANCE GENE COMPLEXES: Evolution and Utilization

The Xa1 gene in rice confers resistance to Japanese race 1 of Xanthomonas oryzae pv. oryzae, the causal pathogen of bacterial blight (BB). We isolated the Xa1 gene by a map-based cloning strategy. The deduced amino acid sequence of the Xa1 gene product contains nucleotide binding sites (NBS) and a new type of leucine-rich repeats (LRR); thus, Xa1 is a member of the NBS-LRR class of plant disease-resistance genes, but quite different from Xa21, another BB-resistance gene isolated from rice. Interestingly, Xa1 gene expression was induced on inoculation with a bacterial pathogen and wound, unlike other isolated resistance genes in plants, which show constitutive expression. The induced expression may be involved in enhancement of resistance against the pathogen.

https://www.pnas.org/content/pnas/95/4/1663.full.pdf

Expression of Xa1, a bacterial blight-resistance gene in rice, is induced by bacterial inoculation

The Hs1pro-1 locus confers resistance to the beet cyst nematode (Heterodera schachtii Schmidt), a major pest in the cultivation of sugar beet (Beta vulgaris L.). The Hs1pro-1 gene was cloned with the use of genome-specific satellite markers and chromosomal break-point analysis. Expression of the corresponding complementary DNA in a susceptible sugar beet conferred resistance to infection with the beet cyst nematode. The native Hs1pro-1 gene, expressed in roots, encodes a 282-amino acid protein with imperfect leucine-rich repeats and a putative membrane-spanning segment, features similar to those of disease resistance genes previously cloned from higher plants.

https://science.sciencemag.org/content/sci/275/5301/832.full.pdf

Positional Cloning of a Gene for Nematode Resistance in Sugar Beet

The Hs1pro-1 gene confers resistance to the beet cyst nematode Heterodera schachtii in sugar beet (Beta vulgaris L) on the basis of a gene-for-gene relationship RNA-gel blot analysis revealed that the transcript of Hs1pro-1 was present in uninfected roots of resistant beet at low levels but increased by about fourfold one day after nematode infection Treatments of plants with external stimuli including salicylic acid, jasmonic acid, gibberellic acid and abscisic acid as well as wounding or salt stress did not result in changes in the gene transcription, indicating de novo transcription of Hs1pro-1 upon nematode infection specifically To study transcriptional regulation of Hs1pro-1 expression at the cellular level, a 3082 bp genomic fragment representing the Hs1pro-1 promoter, isolated from the YAC-DNA housing the Hs1pro-1 gene, was fused to the beta-glucuronidase reporter gene (1832prm1::GUS) and transformed into susceptible beet roots and Arabidopsis plants, respectively Fluorometric and histochemical GUS assays on transgenic beet roots and Arabidopsis plants carrying the 1832prm1::GUS construct demonstrated that the Hs1pro-1 promoter is functional in both species and drives a nematode responsive and feeding site-specific GUS-expression GUS activity was detected as early as at initiation of the nematode feeding sites and GUS staining was restricted to the nematode feeding sites To delineate the regulatory domains of the Hs1pro-1 promoter, fusion genes with various 5' deletions of the Hs1pro-1 promoter and the GUS gene were constructed and analysed in transgenic beet roots as well Cis elements responsible for feeding site-specific gene expression reside between -355 and +247 from the transcriptional initiation site of Hs1pro-1 whereas an enhancer region necessary for higher gene expression is located between -1199 and -705 of the promoter The Hs1pro-1 promoter drives a nematode feeding site-specific GUS expression in both sugar beet and Arabidopsis suggesting a conserved mechanism of regulation of Hs1pro-1 expression in these two species

The promoter of the nematode resistance gene Hs1pro-1 activates a nematode-responsive and feeding site-specific gene expression in sugar beet (Beta vulgaris L.) and Arabidopsis thaliana.

A transformation protocol, based on co-inoculation with two strains of Agrobacterium, Agrobacterium tumefaciens LBA4404 and A. rhizogenes 15834 containing a binary vector with the GUS gene, was established for the induction of transgenic hairy roots from sugar beet (Beta vulgaris L.) explants. It resulted in marked improvement in the formation of hairy roots and the integration of the binary vector T-DNA into the host genome. Of 250 inoculated sugar beet hypocotyls, 84% yielded hairy roots 5–7 days after inoculation, of which 70% were co-transformed with the binary vector T-DNA. To determine stable expression of alien genes in hairy roots, the nematode resistance gene Hs1

 pro-1
 was used as a reporter gene. In addition, molecular marker analysis was applied to monitor stable incorporation of a translocation from the wild beet B. procumbens. The molecular analysis and the nematode (Heterodera schachtii) resistance test in vitro demonstrated that the genomic structure and the expression of the Hs1

 pro-1
 -mediated nematode resistance were well-maintained in all hairy root cultures even after repeated sub-culture.

An improved transformation protocol for studying gene expression in hairy roots of sugar beet (Beta vulgaris L.)

The beet cyst nematode (BCN, Heterodera schachtii Schm.) is a major pest of sugarbeet (Beta vulgaris L.). Chemical control of the parasite poses a threat to the environment and a wide crop rotation is economically impractical. Therefore, breeding of nematode resistant sugarbeet varieties is a promising alternative. Monogenic resistance has been introduced to cultivated sugarbeet from nematode resistant wild beet of the section Procumbentes by interspecific crosses. Different nematode resistant cytogenetic mutants have been selected from the offspring. However, the agronomic performance of these lines is poor and the transmission of the resistance reduced due to meiotic disturbances. Molecular markers have been developed suitable for marker-assisted selection (MAS) of nematode resistant plants in the breeding process and for the cloning of the BCN resistance gene. The novel positional cloning strategy described here makes use of repetitive wild beet specific DNA elements that are tightly linked to the resistance gene. The next step of the cloning procedure comprises the construction of representative yeast artificial chromosome (YAC) libraries and the subsequent screening with molecular markers to extract specific YAC clones encompassing the resistance locus. Candidate genes have been selected from root specific cDNA libraries using the YACs as probes. To identify the resistance gene a root specific complementation system has been established using Agrobacterium rhizogenes mediated transformation. Finally, transgenic hairy roots can be analysed for the presence of the BCN resistance gene by in vitro nematode testing.

Sirak Kifle

Papers

Positional Cloning of a Gene for Nematode Resistance in Sugar Beet

The promoter of the nematode resistance gene Hs1pro-1 activates a nematode-responsive and feeding site-specific gene expression in sugar beet (Beta vulgaris L.) and Arabidopsis thaliana.

An improved transformation protocol for studying gene expression in hairy roots of sugar beet (Beta vulgaris L.)

Breeding for Nematode Resistance in Sugarbeet: A Molecular Approach