Avirulence (AVR) Gene-Based Diagnosis Complements Existing Pathogen Surveillance Tools for Effective Deployment of Resistance (R) Genes Against Rice Blast Disease.

Avirulence (AVR) genes in Magnaporthe oryzae, the fungal pathogen that causes the devastating rice blast disease, have been documented to be major targets subject to mutations to avoid recognition by resistance (R) genes. In this study, an AVR-gene-based diagnosis tool for determining the virulence spectrum of a rice blast pathogen population was developed and validated. A set of 77 single-spore field isolates was subjected to pathotype analysis using differential lines, each containing a single R gene, and classified into 20 virulent pathotypes, except for 4 isolates that lost pathogenicity. In all, 10 differential lines showed low frequency (<24%) of resistance whereas 8 lines showed a high frequency (>95%), inferring the effectiveness of R genes present in the respective differential lines. In addition, the haplotypes of seven AVR genes were determined by polymerase chain reaction amplification and sequencing, if applicable. The calculated frequency of different AVR genes displayed significant variations in the population. AVRPiz-t and AVR-Pii were detected in 100 and 84.9% of the isolates, respectively. Five AVR genes such as AVR-Pik-D (20.5%) and AVR-Pik-E (1.4%), AVRPiz-t (2.7%), AVR-Pita (0%), AVR-Pia (0%), and AVR1-CO39 (0%) displayed low or even zero frequency. The frequency of AVR genes correlated almost perfectly with the resistance frequency of the cognate R genes in differential lines, except for International Rice Research Institute-bred blast-resistant lines IRBLzt-T, IRBLta-K1, and IRBLkp-K60. Both genetic analysis and molecular marker validation revealed an additional R gene, most likely Pi19 or its allele, in these three differential lines. This can explain the spuriously higher resistance frequency of each target R gene based on conventional pathotyping. This study demonstrates that AVR-gene-based diagnosis provides a precise, R-gene-specific, and differential line-free assessment method that can be used for determining the virulence spectrum of a rice blast pathogen population and for predicting the effectiveness of target R genes in rice varieties.

Assessment of the Virulence Spectrum and Its Association with Genetic Diversity in Magnaporthe oryzae Populations from Sub-Saharan Africa.

A collection of 122 isolates of Magnaporthe oryzae, from nine sub-Saharan African countries, was assessed for virulence diversity and genetic relatedness. The virulence spectrum was assessed by pathotype analysis with a panel of 43 rice genotypes consisting of differential lines carrying 24 blast resistance genes (R-genes), contemporary African rice cultivars, and susceptible checks. The virulence spectrum among isolates ranged from 5 to 80%. Five isolates were avirulent to the entire rice panel, while two isolates were virulent to ∼75% of the panel. Overall, cultivar 75-1-127, the Pi9 R-gene donor, was resistant to all isolates (100%), followed by four African rice cultivars (AR105, NERICA 15, 96%; NERICA 4, 91%; and F6-36, 90%). Genetic relatedness of isolates was assessed by single nucleotide polymorphisms derived from genotyping-by-sequencing and by vegetative compatibility tests. Phylogenetic analysis of SNPs of a subset of isolates (n = 78) revealed seven distinct clades that differed in virulence. Principal component analysis showed isolates from East Africa were genetically distinct from those from West Africa. Vegetative compatibility tests of a subset of isolates (n = 65) showed no common groups among countries. This study shows that blast disease could be controlled by pyramiding of Pi9 together with other promising R-genes into rice cultivars that are adapted to East and West African regions.

Construction and high-throughput phenotypic screening ofZymoseptoria tritici over-expression strains.

Targeted gene deletion has been instrumental in elucidating many aspects of Zymoseptoria tritici pathogenicity. Gene over-expression is a complementary approach that is amenable to rapid strain construction and high-throughput screening, which has not been exploited to analyze Z. tritici, largely due to a lack of available techniques. Here we exploit the Gateway® cloning technology for rapid construction of over-expression vectors and improved homologous integration efficiency of a Z. tritici Δku70 strain to build a pilot over-expression library encompassing 32 genes encoding putative DNA binding proteins, GTPases or kinases. We developed a protocol using a Rotor-HDA robot for rapid and reproducible cell pinning for high-throughput in vitro screening. This screen identified an over-expression strain that demonstrated a marked reduction in hyphal production relative to the isogenic progenitor. This study provides a protocol for rapid generation of Z. tritici over-expression libraries and a technique for functional genomic screening in this important pathogen.

A codon-optimized green fluorescent protein for live cell imaging in Zymoseptoria tritici.

Fluorescent proteins (FPs) are powerful tools to investigate intracellular dynamics and protein localization. Cytoplasmic expression of FPs in fungal pathogens allows greater insight into invasion strategies and the host-pathogen interaction. Detection of their fluorescent signal depends on the right combination of microscopic setup and signal brightness. Slow rates of photo-bleaching are pivotal for in vivo observation of FPs over longer periods of time. Here, we test green-fluorescent proteins, including Aequorea coerulescens GFP (AcGFP), enhanced GFP (eGFP) from Aequorea victoria and a novel Zymoseptoria tritici codon-optimized eGFP (ZtGFP), for their usage in conventional and laser-enhanced epi-fluorescence, and confocal laser-scanning microscopy. We show that eGFP, expressed cytoplasmically in Z. tritici, is significantly brighter and more photo-stable than AcGFP. The codon-optimized ZtGFP performed even better than eGFP, showing significantly slower bleaching and a 20-30% further increase in signal intensity. Heterologous expression of all GFP variants did not affect pathogenicity of Z. tritici. Our data establish ZtGFP as the GFP of choice to investigate intracellular protein dynamics in Z. tritici, but also infection stages of this wheat pathogen inside host tissue.

Exploitation of sulfonylurea resistance marker and non-homologous end joining mutants for functional analysis in Zymoseptoria tritici.

The lack of techniques for rapid assembly of gene deletion vectors, paucity of selectable marker genes available for genetic manipulation and low frequency of homologous recombination are major constraints in construction of gene deletion mutants in Zymoseptoria tritici. To address these issues, we have constructed ternary vectors for Agrobacterium tumefaciens mediated transformation of Z. tritici, which enable the single step assembly of multiple fragments via yeast recombinational cloning. The sulfonylurea resistance gene, which is a mutated allele of the Magnaporthe oryzae ILV2 gene, was established as a new dominant selectable marker for Z. tritici. To increase the frequency of homologous recombination, we have constructed Z. tritici strains deficient in the non-homologous end joining pathway of DNA double stranded break repair by inactivating the KU70 and KU80 genes. Targeted gene deletion frequency increased to more than 85% in both Z. tritici ku70 and ku80 null strains, compared to ⩽10% seen in the wild type parental strain IPO323. The in vitro growth and in planta pathogenicity of the Z. tritici ku70 and ku80 null strains were comparable to strain IPO323. Together these molecular tools add significantly to the platform available for genomic analysis through targeted gene deletion or promoter replacements and will facilitate large-scale functional characterization projects in Z. tritici.

A gene locus for targeted ectopic gene integration in Zymoseptoria tritici.

Understanding the cellular organization and biology of fungal pathogens requires accurate methods for genomic integration of mutant alleles or fluorescent fusion-protein constructs. In Zymoseptoria tritici, this can be achieved by integrating of plasmid DNA randomly into the genome of this wheat pathogen. However, untargeted ectopic integration carries the risk of unwanted side effects, such as altered gene expression, due to targeting regulatory elements, or gene disruption following integration into protein-coding regions of the genome. Here, we establish the succinate dehydrogenase (sdi1) locus as a single "soft-landing" site for targeted ectopic integration of genetic constructs by using a carboxin-resistant sdi1(R) allele, carrying the point-mutation H267L. We use various green and red fluorescent fusion constructs and show that 97% of all transformants integrate correctly into the sdi1 locus as single copies. We also demonstrate that such integration does not affect the pathogenicity of Z. tritici, and thus the sdi1 locus is a useful tool for virulence analysis in genetically modified Z. tritici strains. Furthermore, we have developed a vector which facilitates yeast recombination cloning and thus allows assembly of multiple overlapping DNA fragments in a single cloning step for high throughput vector and strain generation.

Tibiotalocalcaneal arthrodesis with a compressive retrograde nail: A retrospective study of 59 nails.

BACKGROUND: Tibiotalocalcaneal arthrodesis is an important salvage method for patients with complex hindfoot problems. This study reports the elective results of combined subtalar and ankle arthrodesis using one design of retrograde intramedullary compression nail. METHODS: Retrospective review identified 58 patients undergoing 59 tibiotalocalcaneal arthrodesis procedures. Mean follow up was 9.15 (3-36) months with average age 60.7 (22-89) years. A function and subjective patient satisfaction questionnaire was achieved in 89%. RESULTS: 53 patients (93%) achieved union at a mean time of 4.17 months. Four patients (8%) subjectively thought the procedure was of no benefit while 42 (84%) had an excellent or good result. The mean visual analogue scale (VAS) score for preoperative functional pain was 7.46 compared to 1.98 post-operatively (p<0.001). CONCLUSIONS: This device and technique offers an effective treatment of hindfoot pathology giving reliable compression and subsequent fusion with excellent patient satisfaction and pain relief. LEVEL OF EVIDENCE: IV case series.

mRNA translocation and microtubules: insect ovary models.

The nurse cells in insect ovarioles supply the developing oocytes with various cellular components, including mRNAs, which pass from one cell to the other through intercellular bridges traversed by microtubules. Best studied of these mRNAs are those that encode the axis-determining factors in Drosophila embryos. These mRNAs are further translocated and localized within the oocyte to sites where the products of their translation will ultimately function. This article explores the evidence supportive of a role for microtubules and motor proteins in these processes.

Growing into the air.

Fungi must grow into the air for reproduction and spore dispersal, and to do this their hyphae contain morphogenetic proteins that respond to the aerial environment. The recent discovery of 'repellent' proteins, however, suggests fungi have more than one mechanism for aerial development.

MPG1 Encodes a Fungal Hydrophobin Involved in Surface Interactions during Infection-Related Development of Magnaporthe grisea.

The rice blast fungus expresses a pathogenicity gene, MPG1, during appressorium formation, disease symptom development, and conidiation. The MPG1 gene sequence predicts a small protein belonging to a family of fungal proteins designated hydrophobins. Using random ascospore analysis and genetic complementation, we showed that MPG1 is necessary for infection-related development of Magnaporthe grisea on rice leaves and for full pathogenicity toward susceptible rice cultivars. The protein product of MPG1 appears to interact with hydrophobic surfaces, where it may act as a developmental sensor for appressorium formation. Ultrastructural studies revealed that MPG1 directs formation of a rodlet layer on conidia composed of interwoven ~5-nm rodlets, which contributes to their surface hydrophobicity. Using combined genetic and biochemical approaches, we identified a 15-kD secreted protein with characteristics that establish it as a class I hydrophobin. The protein is able to form detergent-insoluble high molecular mass complexes, is soluble in trifluoroacetic acid, and exhibits mobility shifts after treatment with performic acid. The production of this protein is directed by MPG1.

Infection-related development in the rice blast fungus Magnaporthe grisea.

Recent developments have been made in the identification of signal transduction pathways and gene products involved in the infection-related development of the rice blast fungus, Magnaporthe grisea. It has been established that cAMP-dependent and MAP kinase-mediated signaling are both critical for appressorium morphogenesis and function. These signaling pathways may act downstream of hydrophobin-mediated surface sensing by the growing germ tube. Several genes have been identified that are required for invasive growth of M. grisea including genes that allow adaptation of fungal metabolism to growth within plant tissues.

Surface attachment and pre-penetration stage development by plant pathogenic fungi.

Fungal pathogens cause many of the most serious crop diseases. One of the principal reasons for the success of this group is their ability to locate and perceive appropriate host surfaces and then to elaborate specialized infection structures. Here we review the processes implicated in surface attachment, germ tube elongation, and development of appressoria. The involvement of surface-acting proteins such as fungal hydrophobins and integrins in these processes is evaluated, along with a description of studies that have revealed the existence of conserved signaling pathways that regulate appressorium formation. Finally, we anticipate the prospect of genome-level analysis of fungal pathogens and the key research questions that will need to be addressed.

Having a blast: exploring the pathogenicity of Magnaporthe grisea.

The rice blast fungus Magnaporthe grisea has an exquisite level of pathogenic specialization, allowing it to infect and colonize rice, subvert the metabolism of the host and spread to new hosts. Through a combination of cytology and molecular-genetic analysis, a picture is gradually emerging of the many interlinked processes that are required for successful infection of the plant.

Complementation of the Magnaporthe grisea deltacpkA mutation by the Blumeria graminis PKA-c gene: functional genetic analysis of an obligate plant pathogen.

Obligate plant-pathogenic fungi have proved extremely difficult to characterize with molecular genetics because they cannot be cultured away from host plants and only can be manipulated experimentally in limited circumstances. Previously, in order to characterize signal transduction processes during infection-related development of the powdery mildew fungus Blumeria graminis (syn. Erysiphe graminis) f. sp. hordei, we described a gene similar to the catalytic subunit of cyclic AMP-dependent protein kinase A (here renamed Bka1). Functional characterization of this gene has been achieved by expression in a deltacpkA mutant of the nonobligate pathogen Magnaporthe grisea. This nonpathogenic M. grisea deltacpkA mutant displays delayed and incomplete appressorium development, suggesting a role for PKA-c in the signal transduction processes that control the maturation of infection cells. Transformation of the deltacpkA mutant with the mildew Bka1 open reading frame, controlled by the M. grisea MPG1 promoter, restored pathogenicity and appressorium maturation kinetics. The results provide, to our knowledge, the first functional genetic analysis of pathogenicity in an obligate pathogen and highlight the remarkable conservation of signaling components regulating infection-related development in pathogenic fungi.

Expression of reverse transcriptase genes in Fulvia fulva.

Antibodies raised against intercellular fluid antigens isolated from diseased tomato leaves have revealed that the fungal pathogen Fulvia fulva expresses genes for a fungal reverse transcriptase (RNA-dependent DNA polymerase). This enzyme is required for the replication of retroviruses and retroviral-like transposable elements and could provide a mechanism for increasing the mutation rate of fungal pathogens, perhaps explaining their ability to evolve new races rapidly. We report here the DNA sequence of a 225-bp clone from a lambda gt11 genomic library of F. fulva. This clone, designated P5, exhibits a high degree of sequence homology with the reverse transcriptase (pol) gene of the Drosophila melanogaster copia-like retrotransposon 17.6. Southern blot analysis of genomic DNA of F. fulva showed that P5-related sequences are moderately reiterated with 30-100 copies, some of which exhibit restriction fragment length polymorphism in different races of the pathogen. Western blot analysis of extracts from F. fulva with antibodies raised to purified reverse transcriptase (from human immunodeficiency virus-1) revealed immunoreactive proteins. Reverse transcriptase previously has been detected in a variety of organisms including yeast, insects, protozoa, and mammals, but to our knowledge, this is the first report of its occurrence in filamentous fungi.

Magnaporthe oryzae populations adapted to finger millet and rice exhibit distinctive patterns of genetic diversity, sexuality and host interaction.

In this study, host-specific forms of the blast pathogen Magnaporthe oryzae in sub-Saharan Africa (SSA) were characterised from distinct cropping locations using a combination of molecular and biological assays. Finger millet blast populations in East Africa revealed a continuous genetic variation pattern and lack of clonal lineages, with a wide range of haplotypes. M. oryzae populations lacked the grasshopper (grh) element (96%) and appeared distinct to those in Asia. An overall near equal distribution (47-53%) of the mating types MAT1-1 and MAT1-2, high fertility status (84-89%) and the dominance of hermaphrodites (64%) suggest a strong sexual reproductive potential. Differences in pathogen aggressiveness and lack of cultivar incompatibility suggest the importance of quantitative resistance. Rice blast populations in West Africa showed a typical lineage-based structure. Among the nine lineages identified, three comprised ~90% of the isolates. Skewed distribution of the mating types MAT1-1 (29%) and MAT1-2 (71%) was accompanied by low fertility. Clear differences in cultivar compatibility within and between lineages suggest R gene-mediated interactions. Distinctive patterns of genetic diversity, sexual reproductive potential and pathogenicity suggest adaptive divergence of host-specific forms of M. oryzae populations linked to crop domestication and agricultural intensification.

The molecular biology of appressorium turgor generation by the rice blast fungus Magnaporthe grisea.

The rice blast fungus Magnaporthe grisea develops specialized infection structures known as appressoria, which develop enormous turgor pressure to bring about plant infection. Turgor is generated by accumulation of compatible solutes, including glycerol, which is synthesized in large quantities in the appressorium. Glycogen, trehalose and lipids represent the most abundant storage products in M. grisea conidia. Trehalose and glycogen are rapidly degraded during conidial germination and it is known that trehalose synthesis is required for virulence of the fungus. Lipid bodies are transported to the developing appressoria and degraded at the onset of turgor generation, in a process that is cAMP-dependent. A combined biochemical and genetic approach is being used to dissect the process of turgor generation in the rice blast fungus.