Jeff Ellis

The Top 10 fungal pathogens in molecular plant pathology

The aim of this review was to survey all fungal pathologists with an association with the journal Molecular Plant Pathology and ask them to nominate which fungal pathogens they would place in a Top 10 based on scientific/economic importance. The survey generated 495 votes from the international community, and resulted in the generation of a Top 10 fungal plant pathogen list for Molecular Plant Pathology. The Top 10 list includes, in rank order, (1) Magnaporthe oryzae; (2) Botrytis cinerea; (3) Puccinia spp.; (4) Fusarium graminearum; (5) Fusarium oxysporum; (6) Blumeria graminis; (7) Mycosphaerella graminicola; (8) Colletotrichum spp.; (9) Ustilago maydis; (10) Melampsora lini, with honourable mentions for fungi just missing out on the Top 10, including Phakopsora pachyrhizi and Rhizoctonia solani. This article presents a short resumé of each fungus in the Top 10 list and its importance, with the intent of initiating discussion and debate amongst the plant mycology community, as well as laying down a bench-mark. It will be interesting to see in future years how perceptions change and what fungi will comprise any future Top 10.

Structure, function and evolution of plant disease resistance genes.

Gene-for-gene plant disease resistance involves two basic processes: perception of pathogen attack, followed by responses to limit disease. Perception involves receptors with high degrees of specificity for pathogen strains, which are encoded by disease resistance genes. Large repertoires of distantly related resistance (R) genes with diverse recognitional specificities are found within a single plant species. The generation of R-gene polymorphism involves gene duplication, followed by DNA-sequence divergence by point mutation, and by deletion and duplication of intragenic DNA repeats encoding blocks of leucine-rich elements. Recombination between related genes reassorts this variation to further diversify gene sequences. Pathogen pressure selects functional resistance specificities and results in the maintenance of R-gene diversity. Recent genome-sequence data reveal that the NBS-LRR (i.e. nucleotide-binding site-leucine-rich repeat) class of R genes represents as much as 1% of the Arabidopsis genome. Experimental data have shown that the LRR has a role in determination of specificity. Mutation experiments, in which R-gene signaling has been dissociated from specificity in constitutive signal mutants, have provided the potential for non-specific resistance to be expressed from pathogen-infection-induced promoters in transgenic plants.

Molecular Characterization of the Maize Rp1-D Rust Resistance Haplotype and Its Mutants

The Rp1-D gene for resistance to maize common rust (Puccinia sorghi) is a member of a complex locus (haplotype) composed of Rp1-D and approximately eight other gene homologs. The identity of Rp1-D was demonstrated by using two independent gene-tagging approaches with the transposons Mutator and Dissociation. PIC20, a disease resistance (R) gene analog probe previously mapped to the rp1 locus, detected insertion of Dissociation in an Rp1-D mutation and excision in three revertants. Independent libraries probed with the PIC20 or Mutator probes resulted in isolation of the same gene sequence. Rp1-D belongs to the nucleotide binding site, leucine-rich repeat class of R genes. However, unlike the rust resistance genes M and L6 from flax, the maize Rp1-D gene does not encode an N-terminal domain with similarity to the signal transduction domains of the Drosophila Toll protein and mammalian interleukin-1 receptor. Although the abundance of transcripts of genes from the rp1 complex changed with leaf age, there was no evidence of any change due to inoculation with avirulent or virulent rust biotypes. A set of 27 Rp1-D mutants displayed at least nine different deletions of Rp1-D gene family members that were consistent with unequal crossing-over events. One mutation (Rp1-D*-24) resulted in deletion of all but one gene family member. Other unique deletions were observed in the disease lesion mimic Rp1-D*-21 and the partially susceptible mutant Rp1-D*-5. Different rp1 specificities have distinct DNA fingerprints (haplotypes). Analysis of recombinants between rp1 specificities indicated that recombination had occurred within the rp1 gene complex. Similar analyses indicated that the rust R genes at the rp5 locus, 2 centimorgans distal to rp1, are not closely related to Rp1-D.

The isolation and mapping of disease resistance gene analogs in maize

Many of the plant disease resistance genes that have been isolated encode proteins with a putative nucleotide binding site and leucine-rich repeats (NBS-LRR resistance genes). Oligonucleotide primers based on conserved motifs in and around the NBS of known NBS-LRR resistance proteins were used to amplify sequences from maize genomic DNA by polymerase chain reaction (PCR). Eleven classes of non-cross-hybridizing sequences were obtained that had predicted products with high levels of amino acid identity to NBS-LRR resistance proteins. These maize resistance gene analogs (RGAs) and one RGA clone obtained previously from wheat were used as probes to map 20 restriction fragment length polymorphism (RFLP) loci in maize. Some RFLPs were shown to map to genomic regions containing virus and fungus resistance genes. Perfect cosegregation was observed between RGA loci and the rust resistance loci rp1 and rp3. The RGA probe associated with rp1 also detected deletion events in several rp1 mutants. These data strongly suggest that some of the RGA clones may hybridize to resistance genes.

Maize Adh-1 promoter sequences control anaerobic regulation: addition of upstream promoter elements from constitutive genes is necessary for expression in tobacco.

The promoter region of a maize alcohol dehydrogenase gene (Adh‐1) was linked to a reporter gene encoding chloramphenicol acetyl transferase (CAT) and transformed stably into tobacco cells using T‐DNA vectors. No CAT enzyme activity could be detected in transgenic tobacco plants unless upstream promoter elements from the octopine synthase gene or the cauliflower mosaic virus 35S promoter were supplied in addition to the maize promoter region. CAT enzyme activity and transcription of the chimaeric gene were then readily detected after anaerobic induction. The first 247 bp upstream of the translation initiation codon of the maize Adh‐1 gene were sufficient to impose anaerobic regulation on the hybrid gene and S1 nuclease mapping confirmed mRNA initiation is from the normal maize Adh‐1 transcription start point.

The generation of plant disease resistance gene specificities.

We are gaining an understanding of the molecular basis of resistance specificity and of the natural processes that generate different specificities. This is a prerequisite for the genetic engineering of new plant disease-resistance genes to control diseases for which naturally occurring resistance is inadequate. DNA sequence analysis indicates that point mutation, recombination and selection can generate and maintain the high levels of polymorphism observed in resistance genes. Comparisons of closely related resistance proteins indicate that specificity can be determined by variation in at least two regions. One of these contains leucine-rich repeats, which are a common feature of most resistance proteins.

Identification and mapping of molecular markers linked to rust resistance genes located on chromosome 1RS of rye using wheat-rye translocation lines

Abstract.The short arm of rye (Secale cereale) chromosome 1 has been widely used in breeding programs to incorporate new disease resistance genes into wheat. Using wheat-rye translocation and recombinant lines, molecular markers were isolated and mapped within chromosomal regions of 1RS carrying rust resistance genes Lr26, Sr31, Yr9 from Petkus and SrR from Imperial rye. RFLP markers previously mapped to chromosome 1HS of barley – flanking the complex Mla powdery mildew resistance gene locus – and chromosome 1DS of Aegilops tauschii – flanking the Sr33 stem rust resistance gene – were shown to map on either side of rust resistance genes on 1RS. Three non cross-hybridising Resistance Gene Analog markers, one of them being derived from the Mla gene family, were mapped within same region of 1RS. PCR-based markers were developed which were tightly linked to the rust resistance genes in Imperial and Petkus rye and which have potential for use in marker-assisted breeding.

Structure and function of proteins controlling strain-specific pathogen resistance in plants

Recently recognised structural and amino acid sequence similarities between plant disease resistance (R) proteins and animal proteins such as Apaf-1 and CED-4 are providing conceptual models for resistance protein function. Data from extensive DNA sequencing of resistance gene families are indicating that the leucine-rich repeat motif is an important determinant of gene-for-gene specificity and that intergenic DNA sequence exchange is a major contributor to R gene diversity.

Development of PCR markers for the selection of wheat stem rust resistance genes Sr24 and Sr26 in diverse wheat germplasm

The use of major resistance genes is the most cost-effective strategy for preventing stem rust epidemics in Australian wheat crops. The long-term success of this strategy is dependent on combining resistance genes that are effective against all predominant races of the pathogen, a task greatly assisted by the use of molecular markers linked to individual resistance genes. The wheat stem rust resistance genes Sr24 and Sr26 (derived from Agropyron elongatum) and SrR and Sr31 (derived from rye) are available in wheat as segments of alien chromosome translocated to wheat chromosomes. Each of these genes provides resistance to all races of wheat stem rust currently found in Australia .We have developed robust PCR markers for Sr24 and Sr26 (this study) and SrR and Sr31 (previously reported) that are applicable across a wide selection of Australian wheat germplasm. Wheat lines have recently become available in which the size of the alien segments containing Sr26, SrR and Sr31 has been reduced. Newly developed PCR-markers can be used to identify the presence of the shorter alien segment in all cases. Assuming that these genes have different gene-for-gene specificities and that the wheat industry will discourage the use of varieties carrying single genes only, the newly developed PCR markers will facilitate the incorporation of two or more of the genes Sr24, Sr26, SrR and Sr31 into wheat lines and have the potential to provide durable control to stem rust in Australia and elsewhere.

The ocs-element is a component of the promoters of several T-DNA and plant viral genes

The ocs‐element is an enhancer element first identified in the promoter of the octopine synthase gene (OCS) where it occurs as a 16 bp palindromic sequence. The transcriptional enhancing activity of the ocs‐element correlated with in vitro binding of a transcription factor. We have now identified ocs‐elements in the promoter regions of six other T‐DNA genes involved in opine synthesis and three plant viral promoters including the 35S promoter of cauliflower mosaic virus. These elements bind the ocs transcription factor in vitro and enhance transcription in plant cells. Comparison of the sequences of these 10 elements has defined a 20 bp consensus sequence, TGACG(T/C)AAG(C/G)(G/A)(A/C)T(G/T)ACG(T/C)(A/C)(A/C), which includes the 16 bp palindrome in its central region. We propose the name ocs‐element for this class of promoter elements of similar sequence and function.