Christopher J. Ridout

Genome Expansion and Gene Loss in Powdery Mildew Fungi Reveal Tradeoffs in Extreme Parasitism

From Blight to Powdery Mildew Pathogenic effects of microbes on plants have widespread consequences. Witness, for example, the cultural upheavals driven by potato blight in the 1800s. A variety of microbial pathogens continue to afflict crop plants today, driving both loss of yield and incurring the increased costs of control mechanisms. Now, four reports analyze microbial genomes in order to understand better how plant pathogens function (see the Perspective by Dodds). Raffaele et al. (p. 1540) describe how the genome of the potato blight pathogen accommodates transfer to different hosts. Spanu et al. (p. 1543) analyze what it takes to be an obligate biotroph in barley powdery mildew, and Baxter et al. (p. 1549) ask a similar question for a natural pathogen of Arabidopsis. Schirawski et al. (p. 1546) compared genomes of maize pathogens to identify virulence determinants. Better knowledge of what in a genome makes a pathogen efficient and deadly is likely to be useful for improving agricultural crop management and breeding. A group of papers analyzes pathogen genomes to find the roots of virulence, opportunism, and life-style determinants. Powdery mildews are phytopathogens whose growth and reproduction are entirely dependent on living plant cells. The molecular basis of this life-style, obligate biotrophy, remains unknown. We present the genome analysis of barley powdery mildew, Blumeria graminis f.sp. hordei (Blumeria), as well as a comparison with the analysis of two powdery mildews pathogenic on dicotyledonous plants. These genomes display massive retrotransposon proliferation, genome-size expansion, and gene losses. The missing genes encode enzymes of primary and secondary metabolism, carbohydrate-active enzymes, and transporters, probably reflecting their redundancy in an exclusively biotrophic life-style. Among the 248 candidate effectors of pathogenesis identified in the Blumeria genome, very few (less than 10) define a core set conserved in all three mildews, suggesting that most effectors represent species-specific adaptations.

HIGS: Host-Induced Gene Silencing in the Obligate Biotrophic Fungal Pathogen Blumeria graminis

This work examines the effects of RNA interference constructs expressed in host cells on target RNAs in Blumeria graminis, an obligate biotrophic fungal pathogen of barley, and finds that RNAs in the host can affect pathogen transcript levels and pathogen development, thereby providing both a useful research tool and a potentially important means for engineering plant disease resistance. Powdery mildew fungi are obligate biotrophic pathogens that only grow on living hosts and cause damage in thousands of plant species. Despite their agronomical importance, little direct functional evidence for genes of pathogenicity and virulence is currently available because mutagenesis and transformation protocols are lacking. Here, we show that the accumulation in barley (Hordeum vulgare) and wheat (Triticum aestivum) of double-stranded or antisense RNA targeting fungal transcripts affects the development of the powdery mildew fungus Blumeria graminis. Proof of concept for host-induced gene silencing was obtained by silencing the effector gene Avra10, which resulted in reduced fungal development in the absence, but not in the presence, of the matching resistance gene Mla10. The fungus could be rescued from the silencing of Avra10 by the transient expression of a synthetic gene that was resistant to RNA interference (RNAi) due to silent point mutations. The results suggest traffic of RNA molecules from host plants into B. graminis and may lead to an RNAi-based crop protection strategy against fungal pathogens.

Silencing of aphid genes by dsRNA feeding from plants.

Background RNA interference (RNAi) is a valuable reverse genetics tool to study gene function in various organisms, including hemipteran insects such as aphids. Previous work has shown that RNAi-mediated knockdown of pea aphid (Acyrthosiphon pisum) genes can be achieved through direct injection of double-stranded RNA (dsRNA) or small-interfering RNAs (siRNA) into the pea aphid hemolymph or by feeding these insects on artificial diets containing the small RNAs. Methodology/Principal Findings In this study, we have developed the plant-mediated RNAi technology for aphids to allow for gene silencing in the aphid natural environment and minimize handling of these insects during experiments. The green peach aphid M. persicae was selected because it has a broad plant host range that includes the model plants Nicotiana benthamiana and Arabidopsis thaliana for which transgenic materials can relatively quickly be generated. We targeted M. persicae Rack1, which is predominantly expressed in the gut, and M. persicae C002 (MpC002), which is predominantly expressed in the salivary glands. The aphids were fed on N. benthamiana leaf disks transiently producing dsRNA corresponding to these genes and on A. thaliana plants stably producing the dsRNAs. MpC002 and Rack-1 expression were knocked down by up to 60% on transgenic N. benthamiana and A. thaliana. Moreover, silenced M. persicae produced less progeny consistent with these genes having essential functions. Conclusions/Significance Similar levels of gene silencing were achieved in our plant-mediated RNAi approach and published silencing methods for aphids. Furthermore, the N. benthamiana leaf disk assay can be developed into a screen to assess which genes are essential for aphid survival on plants. Our results also demonstrate the feasibility of the plant-mediated RNAi approach for aphid control.

Multiple avirulence paralogues in cereal powdery mildew fungi may contribute to parasite fitness and defeat of plant resistance

Powdery mildews, obligate biotrophic fungal parasites on a wide range of important crops, can be controlled by plant resistance (R) genes, but these are rapidly overcome by parasite mutants evading recognition. It is unknown how this rapid evolution occurs without apparent loss of parasite fitness. R proteins recognize avirulence (AVR) molecules from parasites in a gene-for-gene manner and trigger defense responses. We identify AVRa10 and AVRk1 of barley powdery mildew fungus, Blumeria graminis f sp hordei (Bgh), and show that they induce both cell death and inaccessibility when transiently expressed in Mla10 and Mlk1 barley (Hordeum vulgare) varieties, respectively. In contrast with other reported fungal AVR genes, AVRa10 and AVRk1 encode proteins that lack secretion signal peptides and enhance infection success on susceptible host plant cells. AVRa10 and AVRk1 belong to a large family with >30 paralogues in the genome of Bgh, and homologous sequences are present in other formae speciales of the fungus infecting other grasses. Our findings imply that the mildew fungus has a repertoire of AVR genes, which may function as effectors and contribute to parasite virulence. Multiple copies of related but distinct AVR effector paralogues might enable populations of Bgh to rapidly overcome host R genes while maintaining virulence.

Plant–pathogen interactions: disease resistance in modern agriculture

The growing human population will require a significant increase in agricultural production. This challenge is made more difficult by the fact that changes in the climatic and environmental conditions under which crops are grown have resulted in the appearance of new diseases, whereas genetic changes within the pathogen have resulted in the loss of previously effective sources of resistance. To help meet this challenge, advanced genetic and statistical methods of analysis have been used to identify new resistance genes through global screens, and studies of plant-pathogen interactions have been undertaken to uncover the mechanisms by which disease resistance is achieved. The informed deployment of major, race-specific and partial, race-nonspecific resistance, either by conventional breeding or transgenic approaches, will enable the production of crop varieties with effective resistance without impacting on other agronomically important crop traits. Here, we review these recent advances and progress towards the ultimate goal of developing disease-resistant crops.

Coevolution between a family of parasite virulence effectors and a class of LINE-1 retrotransposons.

Parasites are able to evolve rapidly and overcome host defense mechanisms, but the molecular basis of this adaptation is poorly understood. Powdery mildew fungi (Erysiphales, Ascomycota) are obligate biotrophic parasites infecting nearly 10,000 plant genera. They obtain their nutrients from host plants through specialized feeding structures known as haustoria. We previously identified the AVR k1 powdery mildew-specific gene family encoding effectors that contribute to the successful establishment of haustoria. Here, we report the extensive proliferation of the AVR k1 gene family throughout the genome of B. graminis, with sequences diverging in formae speciales adapted to infect different hosts. Also, importantly, we have discovered that the effectors have coevolved with a particular family of LINE-1 retrotransposons, named TE1a. The coevolution of these two entities indicates a mutual benefit to the association, which could ultimately contribute to parasite adaptation and success. We propose that the association would benefit 1) the powdery mildew fungus, by providing a mechanism for amplifying and diversifying effectors and 2) the associated retrotransposons, by providing a basis for their maintenance through selection in the fungal genome.

Use of AFLP in cereals research

Bands of interest can be excised from gels that have been dried onto 3MM filter paper (Whatman) or from dried silver stained gels17xCloning and mapping of variety-specific rice genomic DNA sequences: amplified fragment length polymorphisms (AFLP) from silver stained polyacrylamide gels. Cho, Y.G. et al. Genome. 1996; 39: 373–378Crossref | PubMedSee all References17, and re-hydrated overnight at 4°C. The DNA can then be re-amplified with the original primers and used as a probe, for direct sequencing or for cloning. Also, the AFLP technique can be modified so that one primer is obtained from a known sequence to detect sequence-specific amplification polymorphisms (S-SAP). This approach has been used successfully to map Bare-1 transposable elements throughout the barley genome18xGenetic distribution of Bare-1-like retrotransposable elements in the barley genome revealed by sequence-specific amplification polymorphisms (S-SAP). Waugh, R. et al. Mol. Gen. Genet. 1997; 253: 687–694Crossref | PubMed | Scopus (330)See all References18.Increasingly, AFLP is being used for several specialist applications to assist the rapid isolation and characterization of interesting genes. AFLP-based mRNA fingerprinting has been used to isolate sequences mapping to deleted chromosome segments in cereals or other species19xAFLP-based mRNA fingerprinting. Money, T. et al. Nucleic Acids Res. 1996; 24: 2616–2617Crossref | PubMed | Scopus (68)See all References19 and cDNA-AFLP has been used to monitor the expression of genes20xVisualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: Analysis of gene expression during potato tuber expression. Bachem, C.W.B. et al. Plant J. 1996; 9: 745–753Crossref | PubMedSee all References20. AFLP is also being used to screen superpools of plasmid DNA from 384 clones of bacterial artificial chomosomes (BACs; C. Goldstein and M. Dixon, unpublished), enabling the alignment of contigs and the rapid isolation of genes tightly linked to markers identified through high density mapping. The discriminating power of AFLP has also revealed the cryptic genome changes that could occur in transgenic rice plants generated by particle bombardment or intact cell electroporation21xMolecular analysis of the genome of transgenic rice (Oryza sativa L.). Plants produced via particle bombardment or intact cell electroporation. Arencibia, A. et al. Mol. Breed. 1998; 4: 99–109Crossref | Scopus (37)See all References21. Analysis of complex traits has so far been limited by mapping techniques with a low marker index (MI)22xThe comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. Powell, W. et al. Mol. Breed. 1996; 2: 225–238CrossrefSee all References22. With AFLP the MI is more suitable, and AFLP has recently been applied to the analysis of quantitative traits in barley22xThe comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. Powell, W. et al. Mol. Breed. 1996; 2: 225–238CrossrefSee all References22 and rice23xMapping QTLs for submergence tolerance in rice by AFLP analysis and selective genotyping. Nandi, S. et al. Mol. Gen. Genet. 1997; 255: 1–8Crossref | PubMed | Scopus (110)See all References23.One of the major limitations of AFLP is the objective difficulty in identifying allelic variants at a specific locus, which has resulted in the technique being used almost exclusively for dominant markers. Recently, however, new software for image analysis of fluorescent PCR products has been developed by several companies including Keygene and Perkin Elmer. This will probably be developed for use with AFLP, which would enable AFLP to be used for scoring semi-dominant markers.As more maps of a given species are produced by various research groups, methods to integrate the different maps are clearly necessary to ensure that the same marker can be investigated. In a limited comparison within a single laboratory, accurately sized bands were found to be sufficient to identify homologous products between six different barley crosses5xHomology of AFLP products in three mapping populations of barley. Waugh, R. et al. Mol. Gen. Genet. 1997; 255: 311–321Crossref | PubMed | Scopus (140)See all References5. The reliability of this approach might be enhanced if more selective nucleotides were included to improve the discrimination of the polymorphic band. Further ambiguity could be eliminated by obtaining the sequence of the band.In conclusion, AFLP is a robust and reliable technique that has already proved its value in mapping and phylogenetic studies with a range of cereals. Increasingly, the technique is proving invaluable for the analysis of complex traits, for variety identification and for the rapid isolation of important genes. AFLP is rapidly becoming the preferred molecular technique for many types of investigation and will undoubtedly continue to find new applications in many areas of cereals research.Note: The AFLP technique is covered by patents and/or patent applications pending owned by Keygene n.v. Use of the technique for purposes other than research requires a license from Keygene n.v.

The activity of acetyl-CoA carboxylase is not correlated with the rate of lipid synthesis during development of oilseed rape (Brassica napus L.) embryos

Acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) activity has been determined in seed tissues of oilseed rape (Brassica napus L.), pea (Pisum sativum L.) and castor bean (Ricinus communis L.). A new method is described which leads to significantly higher measurable activities of the enzyme in tissue homogenates than previously reported. This method does not involve either Triton X-100 or centrifugation treatments which have been used previously in the study of the enzyme. In the case of oilseed rape the activity was also increased by removal of the testa from the seed. The activity of ACCase was determined throughout the development of oilseed rape embryos. Enzyme activity increased 3.5-fold as the embryo fresh weight increased from 0.3 to 2.0 mg and then reached a plateau at 1.1 nmol malonyl-CoA-min−1 · embryo−1. The main period of lipid accumulation commenced at an embryo fresh weight of 2.3 mg, which was after the plateau in ACCase activity had been reached. Activity of the enzyme declined after an embryo fresh weight of 3.5 mg, which was before lipid accumulation in the embryo had been completed. Comparison of the activity of ACCase and the apparent in-vivo rate of lipid synthesis on an embryo-fresh-weight basis (i.e. nmol malonyl-CoA formed or utilized·min−1·mg−1 fresh weight) revealed that ACCase activity declines relative to the rate of lipid synthesis throughout development. The negative correlation between these two rates is discussed in relation to the role of ACCase in the regulation of accumulation of storage lipid during embryo development.

Enhanced Disease Resistance Caused by BRI1 Mutation Is Conserved Between Brachypodium distachyon and Barley (Hordeum vulgare)

This study investigated the impact of brassinosteroid (BR)-insensitive 1 (BRI1) mutation, the main receptor of BR in both Brachypodium distachyon and barley, on disease resistance against a range of fungal pathogens of cereals exhibiting different trophic lifestyles. Results presented here show that i) disruption of BRI1 has pleiotropic effects on disease resistance in addition to affecting plant development. BR signaling functions antagonistically with mechanisms of disease resistance that are effective against a broad range of cereal pathogens. ii) Disruption of BRI1 results in increased disease resistance against necrotrophic and hemibiotrophic pathogens that exhibit only a marginal asymptomatic phase but has no effect on biotrophic pathogens or those with a prolonged asymptomatic phase, and iii) disruption of BRI1 has a similar effect on disease resistance in B. distachyon and barley, indicating that defense mechanisms are conserved between these species. This work presents the first evidence for conservation of disease resistance mechanisms between the model species B. distachyon and the cereal crop barley and validates B. distachyon for undertaking model-to-crop translation studies of disease resistance.

Methods to Study PAMP-Triggered Immunity in Brassica Species

The first layer of active defense in plants is based on the perception of pathogen-associated molecular patterns (PAMPs) leading to PAMP-triggered immunity (PTI). PTI is increasingly being investigated in crop plants, where it may have potential to provide durable disease resistance in the field. Limiting this work, however, is an absence of reliable bioassays to investigate PAMP responses in some species. Here, we present a series of methods to investigate PTI in Brassica napus. The assays allow measuring early responses such as the oxidative burst, mitogen-activated protein kinase phosphorylation, and PAMP-induced marker gene expression. Illumina-based RNA sequencing analysis produced a genome-wide survey of transcriptional changes upon PAMP treatment seen in both the A and C genomes of the allotetraploid B. napus. Later responses characterized include callose deposition and lignification at the cell wall, seedling growth inhibition, and PAMP-induced resistance to Pseudomonas syringae and Botrytis cinerea. Furthermore, using these assays, we demonstrated substantial variation in PAMP responses within a collection of diverse B. napus cultivars. The assays reported here could have widespread application in B. napus breeding and mapping programs to improve selection for broad-spectrum disease resistance.