Maintenance of genetic variation in plants and pathogens involves complex networks of gene-for-gene interactions

The RPP13 [recognition of Hyaloperonospora arabidopsidis (previously known as Peronospora parasitica )] resistance ( R ) gene in Arabidopsis thaliana exhibits the highest reported level of sequence diversity among known R genes. Consistent with a co-evolutionary model, the matching effector protein ATR13 ( A. thaliana -recognized) from H. arabidopsidis reveals extreme levels of allelic diversity. We isolated 23 new RPP13 sequences from a UK metapopulation, giving a total of 47 when combined with previous studies. We used these in functional studies of the A. thaliana accessions for their resistance response to 16 isolates of H. arabidopsidis . We characterized the molecular basis of recognition by the expression of the corresponding ATR13 genes from these 16 isolates in these host accessions. This allowed the determination of which alleles of RPP13 were responsible for pathogen recognition and whether recognition was dependent on the RPP13/ATR13 combination. Linking our functional studies with phylogenetic analysis, we determined that: (i) the recognition of ATR13 is mediated by alleles in just a single RPP13 clade; (ii) RPP13 alleles in other clades have evolved the ability to detect other pathogen ATR protein(s); and (iii) at least one gene, unlinked to RPP13 in A. thaliana, detects a different subgroup of ATR13 alleles.     

Differential recognition of highly divergent downy mildew avirulence gene alleles by RPP1 resistance genes from two Arabidopsis lines

The perception of downy mildew avirulence (Arabidopsis thaliana Recognized [ATR]) gene products by matching Arabidopsis thaliana resistance (Recognition of Peronospora parasitica [RPP]) gene products triggers localized cell death (a hypersensitive response) in the host plant, and this inhibits pathogen development. The oomycete pathogen, therefore, is under selection pressure to alter the form of these gene products to prevent detection. That the pathogen maintains these genes indicates that they play a positive role in pathogen survival. Despite significant progress in cloning plant RPP genes and characterizing essential plant components of resistance signaling pathways, little progress has been made in identifying the oomycete molecules that trigger them. Concluding a map-based cloning effort, we have identified an avirulence gene, ATR1NdWsB, that is detected by RPP1 from the Arabidopsis accession Niederzenz in the cytoplasm of host plant cells. We report the cloning of six highly divergent alleles of ATR1NdWsB from eight downy mildew isolates and demonstrate that the ATR1NdWsB alleles are differentially recognized by RPP1 genes from two Arabidopsis accessions (Niederzenz and Wassilewskija). RPP1-Nd recognizes a single allele of ATR1NdWsB; RPP1-WsB also detects this allele plus three additional alleles with divergent sequences. The Emco5 isolate expresses an allele of ATR1NdWsB that is recognized by RPP1-WsB, but the isolate evades detection in planta. Although the Cala2 isolate is recognized by RPP1-WsA, the ATR1NdWsB allele from Cala2 is not, demonstrating that RPP1-WsA detects a novel ATR gene product. Cloning of ATR1NdWsB has highlighted the presence of a highly conserved novel amino acid motif in avirulence proteins from three different oomycetes. The presence of the motif in additional secreted proteins from plant pathogenic oomycetes and its similarity to a host-targeting signal from malaria parasites suggest a conserved role in pathogenicity.     

Structural elucidation and functional characterization of the Hyaloperonospora arabidopsidis effector protein ATR13

The oomycete Hyaloperonospora arabidopsidis (Hpa) is the causal agent of downy mildew on the model plant Arabidopsis thaliana and has been adapted as a model system to investigate pathogen virulence strategies and plant disease resistance mechanisms. Recognition of Hpa infection occurs when plant resistance proteins (R-genes) detect the presence or activity of pathogen-derived protein effectors delivered to the plant host. This study examines the Hpa effector ATR13 Emco5 and its recognition by RPP13-Nd, the cognate R-gene that triggers programmed cell death (HR) in the presence of recognized ATR13 variants. Herein, we use NMR to solve the backbone structure of ATR13 Emco5, revealing both a helical domain and a disordered internal loop. Additionally, we use site-directed and random mutagenesis to identify several amino acid residues involved in the recognition response conferred by RPP13-Nd. Using our structure as a scaffold, we map these residues to one of two surface-exposed patches of residues under diversifying selection. Exploring possible roles of the disordered region within the ATR13 structure, we perform domain swapping experiments and identify a peptide sequence involved in nucleolar localization. We conclude that ATR13 is a highly dynamic protein with no clear structural homologues that contains two surface-exposed patches of polymorphism, only one of which is involved in RPP13-Nd recognition specificity.     

RPP13 is a simple locus in Arabidopsis thaliana for alleles that specify downy mildew resistance to different avirulence determinants in Peronospora parasitica

Disease resistance (R) genes are found in plants as either simple (single allelic series) loci, or more frequently as complex loci of tandemly repeated genes. These different loci are likely to be under similar evolutionary forces from pathogens, but the contrast between them suggests important differences in mechanisms associated with DNA structure and recombination that generate and maintain R gene diversity. The RPP13 locus in Arabidopsis represents an important paradigm for studying the evolution of an R gene at a simple locus. The RPP13 allele from the accession Nd-1, designated RPP13-Nd, confers resistance to five different isolates of the biotrophic oomycete, Peronospora parasitica (causal agent of downy mildew), and encodes an NBS-LRR type R protein with a putative amino-terminal leucine zipper. The RPP13-Rld allele, cloned from the accession Rld-2, encodes a different specificity. Comparison of three RPP13 alleles revealed a high rate of amino acid divergence within the LRR domain, less than 80% identity overall, compared to the remainder of the protein (> 95% identity). We also found evidence for positive selection in the LRR domain for amino acid diversification outside the core conserved beta-strand/beta-turn motif, suggesting that more of the LRR structure is available for interaction with target molecules than has previously been reported for other R gene products. Furthermore, an amino acid sequence (LLRVLDL) identical in an LRR among RPP13 alleles is conserved in other LZ NBS-LRR type R proteins, suggesting functional significance.     

Pronounced intraspecific haplotype divergence at the RPP5 complex disease resistance locus of Arabidopsis

In Arabidopsis ecotype Landsberg erecta (Ler), RPP5 confers resistance to the pathogen Peronospora parasitica. RPP5 is part of a clustered multigene family encoding nucleotide binding-leucine-rich repeat (LRR) proteins. We compared 95 kb of DNA sequence carrying the Ler RPP5 haplotype with the corresponding 90 kb of Arabidopsis ecotype Columbia (Col-0). Relative to the remainder of the genome, the Ler and Col-0 RPP5 haplotypes exhibit remarkable intraspecific polymorphism. The RPP5 gene family probably evolved by extensive recombination between LRRs from an RPP5-like progenitor that carried only eight LRRs. Most members have variable LRR configurations and encode different numbers of LRRs. Although many members carry retroelement insertions or frameshift mutations, codon usage analysis suggests that regions of the genes have been subject to purifying or diversifying selection, indicating that these genes were, or are, functional. The RPP5 haplotypes thus carry dynamic gene clusters with the potential to adapt rapidly to novel pathogen variants by gene duplication and modification of recognition capacity. We propose that the extremely high level of polymorphism at this complex resistance locus is maintained by frequency-dependent selection.     

Frequent sequence exchanges between homologs of RPP8 in Arabidopsis are not necessarily associated with genomic proximity

Disease resistance (R) genes are often clustered in plant genomes and may exhibit heterogeneous rates of evolution. Some (type I R genes) have evolved rapidly through frequent sequence exchanges, while others (type II R genes) have evolved independently and tend to be conserved in different genotypes or related species. The RPP8 resistance gene in Arabidopsis thaliana is located at a complex locus that also harbors the sequence-related resistance genes HRT and RCY1 in different ecotypes. We sequenced 98 homologs of RPP8 from A. thaliana, Arabidopsis arenosa and Arabidopsis lyrata. Three lineages of type II and one lineage of type I RPP8 homologs were identified. Two of the three lineages of type II genes are each represented by a single-copy locus on either chromosomes I or V. Chromosome V contains two small clusters of RPP8 paralogs. One cluster contains both type I and type II genes and the other comprises only type I genes. These multi-copy loci have expanded and contracted through unequal crossovers, which have generated chimeric genes as well as variations in copy number. Sequence exchanges, most likely gene conversions, were detected between RPP8 homologs that are spatially separated by 2.2 Mb and 12 cM. The sequence exchanges between type I homologs within a locus have been more frequent than sequence exchanges between homologs from two different loci, indicating the influence of chromosomal position on the evolution of these R genes. However, physical distance was not the only factor determining the frequency of sequence exchange, because some closely linked paralogs exhibited little sequence exchange. At least two distinct lineages of type II RPP8 homologs were identified in different species, with obvious allelic/orthologous relationships within each lineage. Therefore, the differentiation of type I and type II RPP8 homologs seems to have occurred before speciation of A. thaliana, A. arenosa and A. lyrata.     

Neutral drift and polymorphism in gene-for-gene systems

Pathogens are a main driving force of the evolution of plants and animals. Being resistant to diseases confers a high selective advantage to hosts, yet many host–pathogen systems show a remarkable degree of polymorphism of host resistance and pathogen virulence. The most common explanation of this phenomenon is that both resistance and virulence genes are costly and that there is selection against those genes when they are unnecessary. Here, we use stochastic multi‐locus simulations to show that the origin and the maintenance of genetic polymorphism in plant–pathogen systems can be explained without costs. In multi‐locus gene‐for‐gene systems, temporal domination of a super pathogen can cause polymorphism in resistance through neutral drift. With an increasing number of susceptible alleles in the host population, pathogen types other than the super race are able to cause infections and invade the population, leading to higher pathogen diversity and in turn to higher host diversity.     

Natural variation reveals key amino acids in a downy mildew effector that alters recognition specificity by an Arabidopsis resistance gene

RPP13, a member of the cytoplasmic class of disease resistance genes, encodes one of the most variable Arabidopsis proteins so far identified. This variability is matched in ATR13, the protein from the oomycete downy mildew pathogen Hyaloperonospora parasitica recognized by RPP13, suggesting that these proteins are involved in tight reciprocal coevolution. ATR13 exhibits five domains: an N-terminal signal peptide, an RXLR motif, a heptad leucine/isoleucine repeat, an 11-amino-acid repeated sequence and a C-terminal domain. We show that the conserved RXLR-containing domain is dispensable for ATR13-mediated recognition, consistent with its role in transport into the plant cytoplasm. Sequencing ATR13 from 16 isolates of H. parasitica revealed high levels of amino acid diversity across the entire protein. The leucines/isoleucines of the heptad leucine repeat were conserved, and mutation of particular leucine or isoleucine residues altered recognition by RPP13. Natural variation has not exploited this route to detection avoidance, suggesting a key role of this domain in pathogenicity. The extensive variation in the 11-amino-acid repeat units did not affect RPP13 recognition. Domain swap analysis showed that recognition specificity lay in the C-terminal domain of ATR13. Variation analyses combined with functional assays allowed the identification of four amino acid positions that may play a role in recognition specificity. Site-directed mutagenesis confirmed that a threonine residue is absolutely required for RPP13 recognition and that recognition can be modulated by the presence of either an arginine or glutamic acid at other sites. Mutations in these three amino acids had no effect on the interaction of ATR13 with a resistance gene unlinked to RPP13, consistent with their critical role in determining RPP13-Nd recognition specificity.     

Computational prediction and molecular characterization of an oomycete effector and the cognate Arabidopsis resistance gene

Hyaloperonospora arabidopsidis (Hpa) is an obligate biotroph oomycete pathogen of the model plant Arabidopsis thaliana and contains a large set of effector proteins that are translocated to the host to exert virulence functions or trigger immune responses. These effectors are characterized by conserved amino-terminal translocation sequences and highly divergent carboxyl-terminal functional domains. The availability of the Hpa genome sequence allowed the computational prediction of effectors and the development of effector delivery systems enabled validation of the predicted effectors in Arabidopsis. In this study, we identified a novel effector ATR39-1 by computational methods, which was found to trigger a resistance response in the Arabidopsis ecotype Weiningen (Wei-0). The allelic variant of this effector, ATR39-2, is not recognized, and two amino acid residues were identified and shown to be critical for this loss of recognition. The resistance protein responsible for recognition of the ATR39-1 effector in Arabidopsis is RPP39 and was identified by map-based cloning. RPP39 is a member of the CC-NBS-LRR family of resistance proteins and requires the signaling gene NDR1 for full activity. Recognition of ATR39-1 in Wei-0 does not inhibit growth of Hpa strains expressing the effector, suggesting complex mechanisms of pathogen evasion of recognition, and is similar to what has been shown in several other cases of plant-oomycete interactions. Identification of this resistance gene/effector pair adds to our knowledge of plant resistance mechanisms and provides the basis for further functional analyses.     

The downy mildew effector proteins ATR1 and ATR13 promote disease susceptibility in Arabidopsis thaliana

The downy mildew (Hyaloperonospora parasitica) effector proteins ATR1 and ATR13 trigger RPP1-Nd/WsB- and RPP13-Nd-dependent resistance, respectively, in Arabidopsis thaliana. To better understand the functions of these effectors during compatible and incompatible interactions of H. parasitica isolates on Arabidopsis accessions, we developed a novel delivery system using Pseudomonas syringae type III secretion via fusions of ATRs to the N terminus of the P. syringae effector protein, AvrRPS4. ATR1 and ATR13 both triggered the hypersensitive response (HR) and resistance to bacterial pathogens in Arabidopsis carrying RPP1-Nd/WsB or RPP13-Nd, respectively, when delivered from P. syringae pv tomato (Pst) DC3000. In addition, multiple alleles of ATR1 and ATR13 confer enhanced virulence to Pst DC3000 on susceptible Arabidopsis accessions. We conclude that ATR1 and ATR13 positively contribute to pathogen virulence inside host cells. Two ATR13 alleles suppressed bacterial PAMP (for Pathogen-Associated Molecular Patterns)-triggered callose deposition in susceptible Arabidopsis when delivered by DC3000 DeltaCEL mutants. Furthermore, expression of another allele of ATR13 in plant cells suppressed PAMP-triggered reactive oxygen species production in addition to callose deposition. Intriguingly, although Wassilewskija (Ws-0) is highly susceptible to H. parasitica isolate Emco5, ATR13Emco5 when delivered by Pst DC3000 triggered localized immunity, including HR, on Ws-0. We suggest that an additional H. parasitica Emco5 effector might suppress ATR13-triggered immunity.     

Natural variation in the Pto disease resistance gene within species of wild tomato (Lycopersicon). II. Population genetics of Pto

Disease resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) in the host species Lycopersicon esculentum, the cultivated tomato, and the closely related L. pimpinellifolium is triggered by the physical interaction between the protein products of the host resistance (R) gene Pto and the pathogen avirulence genes AvrPto and AvrPtoB. Sequence variation at the Pto locus was surveyed in natural populations of seven species of Lycopersicon to test hypotheses of host-parasite coevolution and functional adaptation of the Pto gene. Pto shows significantly higher nonsynonymous polymorphism than 14 other non-R-gene loci in the same samples of Lycopersicon species, while showing no difference in synonymous polymorphism, suggesting that the maintenance of amino acid polymorphism at this locus is mediated by pathogen selection. Also, a larger proportion of ancestral variation is maintained at Pto as compared to these non-R-gene loci. The frequency spectrum of amino acid polymorphisms known to negatively affect Pto function is skewed toward low frequency compared to amino acid polymorphisms that do not affect function or silent polymorphisms. Therefore, the evolution of Pto appears to be influenced by a mixture of both purifying and balancing selection.     

Genetic and physical mapping of the RPP13 locus, in Arabidopsis, responsible for specific recognition of several Peronospora parasitica (downy mildew) isolates.

Fifteen isolates of the biotrophic oomycete Peronospora parasitica (downy mildew) were obtained from a population of Arabidopsis thaliana plants that established naturally in a garden the previous year. They exhibited phenotypic variation in a set of 12 Arabidopsis accessions that suggested that the parasite population consisted of at least six pathotypes. One isolate, Maks9, elicited an interaction phenotype of flecking necrosis and no sporulation (FN) in the Arabidopsis accession Nd-1, and more extensive pitting necrosis with no sporulation (PN) in the accession Ws-2. RPP13 was designated as the locus for a single dominant resistance gene associated with the resistance in Nd-1 and mapped to an interval of approximately 60 kb on a bacterial artificial chromosome (BAC) contig on the lower arm of chromosome 3. This locus is approximately 6 cM telomeric to RPP1, which was previously described as the locus for the PN interaction with five Peronospora isolates, including resistance to Maks9 in Ws-2. New Peronospora isolates were obtained from four other geographically distinct populations of P. parasitica. Four isolates were characterized that elicited an FN phenotype in Nd-1 and mapped resistance to the RPP13 locus. This suggests that the RPP13 locus contains either a single gene capable of multiple isolate recognition or a group of tightly linked genes. Further analysis suggests that the RPP11 gene in the accession Rld-0 may be allelic to RPP13 but results in a different recognition capability.     

Natural selection for polymorphism in the disease resistance gene Rps2 of Arabidopsis thaliana

Pathogen resistance is an ecologically important phenotype increasingly well understood at the molecular genetic level. In this article, we examine levels of avrRpt2-dependent resistance and Rps2 locus DNA sequence variability in a worldwide sample of 27 accessions of Arabidopsis thaliana. The rooted parsimony tree of Rps2 sequences drawn from a diverse set of ecotypes includes a deep bifurcation separating major resistance and susceptibility clades of alleles. We find evidence for selection maintaining these alleles and identify the N-terminal part of the leucine-rich repeat region as a probable target of selection. Additional protein variants are found within the two major clades and correlate well with measurable differences among ecotypes in resistance to the avirulence gene avrRpt2 of the pathogen Pseudomonas syringae. Long-lived polymorphisms have been observed for other resistance genes of A. thaliana; the Rps2 data suggest that the long-term maintenance of phenotypic variation in resistance genes may be a general phenomenon and are consistent with diversifying selection acting in concert with selection to maintain variation.     

Molecular evolution of two linked genes, Est-6 and Sod, in Drosophila melanogaster.

We have obtained 15 sequences of Est-6 from a natural population of Drosophila melanogaster to test whether linkage disequilibrium exists between Est-6 and the closely linked Sod, and whether natural selection may be involved. An early experiment with allozymes had shown linkage disequilibrium between these two loci, while none was detected between other gene pairs. The Sod sequences for the same 15 haplotypes were obtained previously. The two genes exhibit similar levels of nucleotide polymorphism, but the patterns are different. In Est-6, there are nine amino acid replacement polymorphisms, one of which accounts for the S-F allozyme polymorphism. In Sod, there is only one replacement polymorphism, which corresponds to the S-F allozyme polymorphism. The transversion/transition ratio is more than five times larger in Sod than in Est-6. At the nucleotide level, the S and F alleles of Est-6 make up two allele families that are quite different from each other, while there is relatively little variation within each of them. There are also two families of alleles in Sod, one consisting of a subset of F alleles, and the other consisting of another subset of F alleles, designed F(A), plus all the S alleles. The Sod F(A) and S alleles are completely or nearly identical in nucleotide sequence, except for the replacement mutation that accounts for the allozyme difference. The two allele families have independent evolutionary histories in the two genes. There are traces of statistically significant linkage disequilibrium between the two genes that, we suggest, may have arisen as a consequence of selection favoring one particular sequence at each locus.     

Reproductive protein evolution within and between species: maintenance of divergent ZP3 alleles in Peromyscus

In a variety of animal taxa, proteins involved in reproduction evolve more rapidly than nonreproductive proteins. Most studies of reproductive protein evolution, however, focus on divergence between species, and little is known about differentiation among populations within a species. Here we investigate the molecular population genetics of the protein ZP3 within two Peromyscus species. ZP3 is an egg coat protein involved in primary binding of egg and sperm and is essential for fertilization. We find that amino acid polymorphism in the sperm-combining region of ZP3 is high relative to silent polymorphism in both species of Peromyscus . In addition, while there is geographical structure at a mitochondrial gene ( Cytb ), a nuclear gene ( Lcat ) and eight microsatellite loci, we find no evidence for geographical structure at Zp3 in Peromyscus truei . These patterns are consistent with the maintenance of ZP3 alleles by balancing selection, possibly due to sexual conflict or pathogen resistance. However, we do not find evidence that reinforcement promotes ZP3 diversification; allelic variation in P. truei is similar among populations, including populations allopatric and sympatric with sibling species. In fact, most alleles are present in all populations sampled across P. truei's range. While additional data are needed to identify the precise evolutionary forces responsible for sequence variation in ZP3, our results suggest that in Peromyscus , selection to maintain divergent alleles within species contributes to the pattern of rapid amino acid substitution observed among species.     

Origin and maintenance of a broad-spectrum disease resistance locus in Arabidopsis

The broad-spectrum mildew resistance genes RPW8.1 and RPW8.2 define a unique type of plant disease resistance (R) gene, and so far homologous sequences have been found in Arabidopsis thaliana only, which suggests a recent origin. In addition to RPW8.1 and RPW8.2, the RPW8 locus contains three homologs of RPW8, HR1, HR2, and HR3, which do not contribute to powdery mildew resistance. To investigate whether RPW8 has originated recently, and if so the processes involved, we have isolated and analyzed the syntenic RPW8 loci from Arabidopsis lyrata, and from Brassica rapa and B. oleracea. The A. lyrata locus contains four genes orthologous to HR1, HR2, HR3, and RPW8.2, respectively. Two syntenic loci have been characterized in Brassica; one locus contains three genes and is present in both B. oleracea and B. rapa, and the other locus contains a single gene and is detected in B. rapa only. The Brassica homologs have highest similarity to HR3. Sequence analyses suggested that the RPW8 gene family in Brassicaceae originated from an HR3-like ancestor gene through a series of duplications and that RPW8.1 and RPW8.2 evolved from functional diversification through positive selection several MYA. Examination of the sequence polymorphism of 32 A. thaliana accessions at the RPW8 locus and their disease reaction phenotypes revealed that the polymorphic RPW8 locus defines a major source of resistance to powdery mildew diseases. A possible evolutionary mechanism by which functional polymorphism at the AtRPW8 locus has been maintained in contemporary populations of A. thaliana is discussed.     

Coevolutionary genetics of plants and pathogens

Summary The genetic polymorphism maintained by host-pathogen coevolution is analysed in a multilocus model. The model assumes gene-for-gene interactions of the type commonly observed between host plants and their fungal pathogens. Unstable (epidemic) systems maintain more resistance genes, fewer virulence genes, and less overall genetic diversity than stable (endemic) diseases. The stability of the system depends primarily on demographic parameters, such as the pathogen's intrinsic rate of increase, rather than genetic parameters, such as the costs of resistance and virulence. At equilibrium the model predicts that the number of resistance alleles in each host plant follows a binomial distribution that depends on the cost to the pathogen for carrying virulence alleles. Similarly, the number of virulence alleles in each pathogen spore follows a binomial distribution that depends on one minus the cost to the host for carrying resistance alleles. Data from wild populations match the predicted binomial distributions.     

Arabidopsis downy mildew resistance gene RPP27 encodes a receptor-like protein similar to CLAVATA2 and tomato Cf-9

The Arabidopsis Ler-RPP27 gene confers AtSgt1b-independent resistance to downy mildew (Peronospora parasitica) isolate Hiks1. The RPP27 locus was mapped to a four-bacterial artificial chromosome interval on chromosome 1 from genetic analysis of a cross between the enhanced susceptibility mutant Col-edm1 (Col-sgt1) and Landsberg erecta (Ler-0). A Cf-like candidate gene in this interval was PCR amplified from Ler-0 and transformed into mutant Col-rpp7.1 plants. Homozygous transgenic lines conferred resistance to Hiks1 and at least four Ler-0 avirulent/Columbia-0 (Col-0) virulent isolates of downy mildew pathogen. A full-length RPP27 cDNA was isolated, and analysis of the deduced amino acid sequences showed that the gene encodes a receptor-like protein (RLP) with a distinct domain structure, composed of a signal peptide followed by extracellular Leu-rich repeats, a membrane spanning region, and a short cytoplasmic carboxyl domain. RPP27 is the first RLP-encoding gene to be implicated in disease resistance in Arabidopsis, enabling the deployment of Arabidopsis techniques to investigate the mechanisms of RLP function. Homology searches of the Arabidopsis genome, using the RPP27, Cf-9, and Cf-2 protein sequences as a starting point, identify 59 RLPs, including the already known CLAVATA2 and TOO MANY MOUTHS genes. A combination of sequence and phylogenetic analysis of these predicted RLPs reveals conserved structural features of the family.     

Sequence variation in the cadherin gene of Ostrinia nubilalis: a tool for field monitoring

Toxin-binding proteins of insect midgut epithelial cells are associated with insect resistance to Bacillus thuringiensis (Bt) Cry toxins. A 5378 nt cDNA encoding a 1717 amino acid putative midgut cadherin-like glycoprotein and candidate Cry1Ab toxin-binding protein was characterized from Ostrinia nubilalis. Intraspecific alignment of partial O. nubilalis cadherin gene sequences identified variance within proposed Cry1A toxin binding region 2 (TBR2), 1328IPLQTSILVVT[I/V] N1340, and flanking Cry1A toxin binding region 1 (TBR1), 861DIEIEIIDTNN871. DNA sequence and PCR-RFLP detected single nucleotide polymorphism between cadherin alleles, and pedigree analysis demonstrated Mendelian inheritance. A population sample from Mead, Nebraska showed allelic polymorphism. These assays may be useful for linkage mapping and field surveillance of wild populations and of O. nubilalis.     

Overexpression of OgPAE1 from wild rice confers fungal resistance against Botrytis cinerea

A full-length cDNA of the OgPAE1 gene encoding the alpha5 subunit of the 20S proteasome was isolated from wild rice (Oryza grandiglumis) treated by wounding or with a fungal elicitor. The deduced amino acid sequence of OgPAE1 comprises 237 amino acids (25.99 kDa), and shows 94.5% homology with Arabidopsis thaliana AtPAE1. Expression of OgPAE1 is regulated by defense-related signaling chemicals such as cantharidin, endothall and jasmonic acid. Overexpression of OgPAE1 in A. thaliana leads to resistance to the fungal pathogen Botrytis cinerea by lowering disease rate and size of necrotic lesions, and by less penetration and colonization of fungal hyphae. The results indicate that the 20S proteasome from wild rice is involved in the B. cinerea defense pathway via an as yet undetermined mechanism.