Publications by authors named "Rahim Mehrabi"

44 Publications

Molecular insights into the compatible and incompatible interactions between sugar beet and the beet cyst nematode.

BMC Plant Biol 2020 Oct 22;20(1):483. Epub 2020 Oct 22.

Department of Biotechnology, Ghent University, Coupure Links 653, B-9000, Ghent, Belgium.

Background: Sugar beet (Beta vulgaris subsp. vulgaris) is an economically important crop that provides nearly one third of the global sugar production. The beet cyst nematode (BCN), Heterodera schachtii, causes major yield losses in sugar beet and other crops worldwide. The most effective and economic approach to control this nematode is growing tolerant or resistant cultivars. To identify candidate genes involved in susceptibility and resistance, the transcriptome of sugar beet and BCN in compatible and incompatible interactions at two time points was studied using mRNA-seq.

Results: In the susceptible cultivar, most defense-related genes were induced at 4 dai while suppressed at 10 dai but in the resistant cultivar Nemakill, induction of genes involved in the plant defense response was observed at both time points. In the compatible interaction, alterations in phytohormone-related genes were detected. The effect of exogenous application of Methyl Jasmonate and ET-generator ethephon on susceptible plants was therefore investigated and the results revealed significant reduction in plant susceptibility. Genes putatively involved in the resistance of Nemakill were identified, such as genes involved in phenylpropanoid pathway and genes encoding CYSTM domain-containing proteins, F-box proteins, chitinase, galactono-1,4-lactone dehydrogenase and CASP-like protein. Also, the transcriptome of the BCN was analyzed in infected root samples and several novel potential nematode effector genes were found.

Conclusions: Our data provides detailed insights into the plant and nematode transcriptional changes occurring during compatible and incompatible interactions between sugar beet and BCN. Many important genes playing potential roles in susceptibility or resistance of sugar beet against BCN, as well as some BCN effectors with a potential role as avr proteins were identified. In addition, our findings indicate the effective role of jasmonate and ethylene in enhancing sugar beet defense response against BCN. This research provides new molecular insights into the plant-nematode interactions that can be used to design novel management strategies against BCN.
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http://dx.doi.org/10.1186/s12870-020-02706-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7583174PMC
October 2020

MADS-Box Transcription Factor Is Responsible for Virulence and Development of the Fungal Wheat Pathogen .

Front Microbiol 2020 18;11:1976. Epub 2020 Aug 18.

Wageningen University and Research, Wageningen Plant Research, Wageningen, Netherlands.

is one of the most economically destructive wheat diseases all over the world and is a model fungal plant pathogen within the ascomycetes. In this study, the instrumental role of the gene encoding a MADS-box transcription factor (TF) in the infection process of was functionally characterized as these proteins play critical roles in the global gene regulation required for various developmental and physiological processes. Our infection assays showed that mutants were attenuated in disease development as a 30 and 90% reduction in chloro-necrotic lesions and pycnidia formation, respectively, were observed in plants inoculated with mutant strains demonstrating that is a crucial factor playing a significant role in the late stage of infection corresponding with pycnidial formation. Our expression analysis demonstrated that the transcript level of is induced at 2 and 20 days post-inoculation, coinciding with pycnidial sporulation. In addition, microscopic analyses showed that branch intensity and biomass production were significantly reduced, indicating that impaired pycnidia formation is a result of impaired differentiation and biomass production in the mutants. Furthermore, melanization, a phenomenon required for fruiting body formation, was significantly hampered in mutants as they were not melanized under all tested temperature and media conditions. Overall, our data showed that impaired disease development of the mutants is mainly due to the significant impact of in different cellular processes, including differentiation, branching, fungal biomass production, and melanization, in which identification of downstream genes are of interest to increase our understanding of this pathosystem.
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http://dx.doi.org/10.3389/fmicb.2020.01976DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7461931PMC
August 2020

Whole-genome diversity, population structure and linkage disequilibrium analysis of globally diverse wheat genotypes using genotyping-by-sequencing DArTseq platform.

3 Biotech 2020 Feb 14;10(2):48. Epub 2020 Jan 14.

College of Agriculture, Sanandaj Branch, Islamic Azad University, Sanandaj, Iran.

In this study, 129 wheat genotypes from globally diverse origins were genotyped using DArTseq (SilicoDArT and SNP) markers. After filtering markers for quality-filtering, 14,270 SilicoDArTs and 6484 SNPs were retained and used for genetic diversity, population structure and linkage disequilibrium analyses. The highest number of SilicoDArT and SNP markers mapped on genome A and B compared to genome D. In both marker types, polymorphism information content (PIC) values ranged from 0.1 to 0.5, while > 0.80% of SilicoDArTs and > 0.44% SNPs showed PIC value more than median (0.25%). Un-weighted Neighbor Joining cluster analysis and Bayesian-based model population structure grouped wheat genotypes into three and four clusters, respectively. Principal component analysis and discriminant analysis of principal component results showed highly match with cluster and population structure analysis. Linkage disequilibrium (LD) was more extensive in both marker types, while graphical display of LD decay for both marker types showed that LD declined in the region close to 15 kbp, where -values corresponded to  = 0.16. Overall, our genetic diversity analysis showed high level of variation in studied wheat genotypes, even though there was no relationship between wheat grouping and origins. This might be attributed to admixture level that occurred during long-term natural selection of wheat genotypes in different parts of the world. Highly diverse wheat genotypes used in this study may possess unique genes and are useful sources in breeding programs to improve grain yield and quality.
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http://dx.doi.org/10.1007/s13205-019-2014-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6960278PMC
February 2020

Virulence of Leaf Rust Physiological Races in Iran From 2010 to 2017.

Plant Dis 2020 Feb 18;104(2):363-372. Epub 2019 Dec 18.

Department of Plant Pathology, University of Minnesota, Minneapolis, MN, U.S.A.

The wheat leaf rust fungus, , has widespread geographical distribution in Iran within the Fertile Crescent region of the Middle East where wheat was domesticated and originated. Therefore, it is of great importance to identify the prevalence and distribution of pathotypes in this area. From 2010 to 2017, 241 single-uredinium isolates of . were purified from 175 collections of made from various hosts in 14 provinces of Iran, and they were tested on 20 Thatcher near-isogenic lines carrying single-leaf rust resistance genes. In total, 86 pathotypes were identified, of which the pathotypes FDTTQ, FDKPQ, FDKTQ, and FDTNQ were most prevalent. No virulence for was detected, whereas virulence for was found only on bread wheat in a few provinces in 2016. Only isolates from durum wheat and wild barley were virulent to . Although virulence for , , and was observed in some years, the virulence frequency for these genes was lower than that of the other genes. collections from host plants with different ploidy levels or genetically dissimilar backgrounds were grouped individually according to genetic distance. Based on these results, collections from barley, durum wheat, oat, triticale, and wild barley were different from those of bread wheat.
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http://dx.doi.org/10.1094/PDIS-06-19-1340-REDOI Listing
February 2020

Author Correction: Phosphopantetheinyl transferase (Ppt)-mediated biosynthesis of lysine, but not siderophores or DHN melanin, is required for virulence of Zymoseptoria tritici on wheat.

Sci Rep 2019 Nov 7;9(1):16536. Epub 2019 Nov 7.

BioIntercations and Crop Protection, Rothamsted Research, Harpenden, Hertfordshire, UK.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-019-53309-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838330PMC
November 2019

Whole Genome Diversity, Population Structure, and Linkage Disequilibrium Analysis of Chickpea ( L.) Genotypes Using Genome-Wide DArTseq-Based SNP Markers.

Genes (Basel) 2019 09 4;10(9). Epub 2019 Sep 4.

Department of Agronomy & Plant Breeding, College of Agriculture, Sanandaj Branch, Islamic Azad University, Sanandaj, P.O. Box:618, Iran.

Characterization of genetic diversity, population structure, and linkage disequilibrium is a prerequisite for proper management of breeding programs and conservation of genetic resources. In this study, 186 chickpea genotypes, including advanced "" breeding lines and Iranian landrace "" chickpea genotypes, were genotyped using DArTseq-Based single nucleotide polymorphism (SNP) markers. Out of 3339 SNPs, 1152 markers with known chromosomal position were selected for genome diversity analysis. The number of mapped SNP markers varied from 52 (LG8) to 378 (LG4), with an average of 144 SNPs per linkage group. The chromosome size that was covered by SNPs varied from 16,236.36 kbp (LG8) to 67,923.99 kbp (LG5), while LG4 showed a higher number of SNPs, with an average of 6.56 SNPs per Mbp. Polymorphism information content (PIC) value of SNP markers ranged from 0.05 to 0.50, with an average of 0.32, while the markers on LG4, LG6, and LG8 showed higher mean PIC value than average. Unweighted neighbor joining cluster analysis and Bayesian-based model population structure grouped chickpea genotypes into four distinct clusters. Principal component analysis (PCoA) and discriminant analysis of principal component (DAPC) results were consistent with that of the cluster and population structure analysis. Linkage disequilibrium (LD) was extensive and LD decay in chickpea germplasm was relatively low. A few markers showed r ≥ 0.8, while 2961 pairs of markers showed complete LD (r = 1), and a huge LD block was observed on LG4. High genetic diversity and low kinship value between pairs of genotypes suggest the presence of a high genetic diversity among the studied chickpea genotypes. This study also demonstrates the efficiency of DArTseq-based SNP genotyping for large-scale genome analysis in chickpea. The genotypic markers provided in this study are useful for various association mapping studies when combined with phenotypic data of different traits, such as seed yield, abiotic, and biotic stresses, and therefore can be efficiently used in breeding programs to improve chickpea.
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http://dx.doi.org/10.3390/genes10090676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6770975PMC
September 2019

Pathogenic Diversity of Isolates and Identification of Resistance Sources in Core Collection of Chickpea Germplasm.

Plant Pathol J 2019 Aug 1;35(4):321-329. Epub 2019 Aug 1.

Kordestan Agricultural and Natural Resources Research and Education Center, Agricultural Research, Education and Extension Organization (AREEO), Sanandaj 33623351, Iran.

Ascochyta blight caused by (Pass.) Lab. (Telomorph: ) (Kov.) is one of the most important fungal diseases in chickpea worldwide. Knowledge about pathogen aggressiveness and identification resistance sources to different pathotypes is very useful for proper decisions in breeding programs. In this study, virulence of 32 isolates from different part of Iran were analyzed on seven chickpea differentials and grouped into six races based on 0-9 rating scale and susceptibility/resistant pattern of chickpea differentials. The least and most frequent races were race V and race I, respectively. Race V and VI showed highly virulence on most of differential, while race I showed least aggressiveness. Resistance pattern of 165 chickpea genotypes also were tested against six different races. ANOVA analysis showed high significant difference for isolate, chickpea genotypes and their interactions. Overall chickpea × isolate (race) interactions, 259 resistance responses (disease severity ≤ 4) were identified. Resistance spectra of chickpea genotypes showed more resistance rate to race I (49.70%) and race III (35.15%), while there were no resistance genotypes to race VI. Cluster analysis based on disease severity rate, grouped chickpea genotypes into four distinct clusters. Interactions between isolates or races used in this study, showed the lack of a genotype with complete resistance. Our finding for virulence pattern of and newly identified resistance sources could be considerably important for integration of ascochyta blight resistance genes into chickpea breeding programs and proper decision in future for germplasm conservation and diseases management.
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http://dx.doi.org/10.5423/PPJ.OA.12.2018.0299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6706013PMC
August 2019

Phosphopantetheinyl transferase (Ppt)-mediated biosynthesis of lysine, but not siderophores or DHN melanin, is required for virulence of Zymoseptoria tritici on wheat.

Sci Rep 2018 11 20;8(1):17069. Epub 2018 Nov 20.

BioIntercations and Crop Protection, Rothamsted Research, Harpenden, Hertfordshire, UK.

Zymoseptoria tritici is the causal agent of Septoria tritici blotch (STB) disease of wheat. Z. tritici is an apoplastic fungal pathogen, which does not penetrate plant cells at any stage of infection, and has a long initial period of symptomless leaf colonisation. During this phase it is unclear to what extent the fungus can access host plant nutrients or communicate with plant cells. Several important primary and secondary metabolite pathways in fungi are regulated by the post-translational activator phosphopantetheinyl transferase (Ppt) which provides an essential co-factor for lysine biosynthesis and the activities of non-ribosomal peptide synthases (NRPS) and polyketide synthases (PKS). To investigate the relative importance of lysine biosynthesis, NRPS-based siderophore production and PKS-based DHN melanin biosynthesis, we generated deletion mutants of ZtPpt. The ∆ZtPpt strains were auxotrophic for lysine and iron, non-melanised and non-pathogenic on wheat. Deletion of the three target genes likely affected by ZtPpt loss of function (Aar- lysine; Nrps1-siderophore and Pks1- melanin), highlighted that lysine auxotrophy was the main contributing factor for loss of virulence, with no reduction caused by loss of siderophore production or melanisation. This reveals Ppt, and the lysine biosynthesis pathway, as potential targets for fungicides effective against Z. tritici.
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http://dx.doi.org/10.1038/s41598-018-35223-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6244202PMC
November 2018

Stress and sexual reproduction affect the dynamics of the wheat pathogen effector AvrStb6 and strobilurin resistance.

Nat Genet 2018 03 12;50(3):375-380. Epub 2018 Feb 12.

Laboratory of Phytopathology, Wageningen University and Research, Wageningen, the Netherlands.

Host resistance and fungicide treatments are cornerstones of plant-disease control. Here, we show that these treatments allow sex and modulate parenthood in the fungal wheat pathogen Zymoseptoria tritici. We demonstrate that the Z. tritici-wheat interaction complies with the gene-for-gene model by identifying the effector AvrStb6, which is recognized by the wheat resistance protein Stb6. Recognition triggers host resistance, thus implying removal of avirulent strains from pathogen populations. However, Z. tritici crosses on wheat show that sex occurs even with an avirulent parent, and avirulence alleles are thereby retained in subsequent populations. Crossing fungicide-sensitive and fungicide-resistant isolates under fungicide pressure results in a rapid increase in resistance-allele frequency. Isolates under selection always act as male donors, and thus disease control modulates parenthood. Modeling these observations for agricultural and natural environments reveals extended durability of host resistance and rapid emergence of fungicide resistance. Therefore, fungal sex has major implications for disease control.
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http://dx.doi.org/10.1038/s41588-018-0052-9DOI Listing
March 2018

The ZtVf1 transcription factor regulates development and virulence in the foliar wheat pathogen Zymoseptoria tritici.

Fungal Genet Biol 2017 12 12;109:26-35. Epub 2017 Oct 12.

Laboratory of Phytopathology, Wageningen University and Research, 6700AA Wageningen, The Netherlands; Wageningen University and Research, Wageningen Plant Research, P.O. Box 16, 6700AA Wageningen, The Netherlands.

The dimorphic fungal pathogen, Zymoseptoria tritici undergoes discrete developmental changes to complete its life cycle on wheat. Molecular mechanisms underlying morphogenesis during infection process of Z. tritici are poorly understood. In this study, we have investigated the role of ZtVf1 gene encoding a transcription factor belonging to C-H subfamily. In planta assays revealed that ZtVf1 is required for virulence. Reduced necrotic lesions and low pycnidia density within the lesions resulted in significantly reduced virulence of ZtVf1 mutants. Cytological analysis showed that the impaired virulence of ZtVf1 mutants attributed to reduced penetration and colonization along with hampered pycnidia differentiation. In vitro phenotyping showed that ZtVf1 deletion affects hyphal branching and biomass production suggesting that the reduced tissue colonization by the ZtVf1 mutant might be due to lower hyphal branching and less fungal biomass production. In addition, the majority of infected substomatal cavities by the ZtVf1 mutant filled with compacted mycelia mat that did not differentiate to mature pycnidia indicating that the impaired melanization negatively affected pycnidia formation and maturation. The ZtVf1 might target multiple genes belonging to different cellular processes whose identification is of eminent interest to increase our understanding of this pathosystem. Overall, the data provided in this study indicates that attenuated pathogenicity of ZtVf1 mutant is due to involvement of this gene in the regulation of both early and late stages of infection.
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http://dx.doi.org/10.1016/j.fgb.2017.10.003DOI Listing
December 2017

Karyotype Variability in Plant-Pathogenic Fungi.

Annu Rev Phytopathol 2017 08;55:483-503

Wageningen Plant Research, Wageningen University and Research, 6700AA Wageningen, The Netherlands; email:

Recent advances in genetic and molecular technologies gradually paved the way for the transition from traditional fungal karyotyping to more comprehensive chromosome biology studies. Extensive chromosomal polymorphisms largely resulting from chromosomal rearrangements (CRs) are widely documented in fungal genomes. These extraordinary CRs in fungi generate substantial genome plasticity compared to other eukaryotic organisms. Here, we review the most recent findings on fungal CRs and their underlying mechanisms and discuss the functional consequences of CRs for adaptation, fungal evolution, host range, and pathogenicity of fungal plant pathogens in the context of chromosome biology. In addition to a complement of permanent chromosomes called core chromosomes, the genomes of many fungal pathogens comprise distinct unstable chromosomes called dispensable chromosomes (DCs) that also contribute to chromosome polymorphisms. Compared to the core chromosomes, the structural features of DCs usually differ for gene density, GC content, housekeeping genes, and recombination frequency. Despite their dispensability for normal growth and development, DCs have important biological roles with respect to pathogenicity in some fungi but not in others. Therefore, their evolutionary origin is also reviewed in relation to overall fungal physiology and pathogenicity.
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http://dx.doi.org/10.1146/annurev-phyto-080615-095928DOI Listing
August 2017

Combating a Global Threat to a Clonal Crop: Banana Black Sigatoka Pathogen Pseudocercospora fijiensis (Synonym Mycosphaerella fijiensis) Genomes Reveal Clues for Disease Control.

PLoS Genet 2016 08 11;12(8):e1005876. Epub 2016 Aug 11.

Plant Research International, Wageningen University and Research, Wageningen, The Netherlands.

Black Sigatoka or black leaf streak disease, caused by the Dothideomycete fungus Pseudocercospora fijiensis (previously: Mycosphaerella fijiensis), is the most significant foliar disease of banana worldwide. Due to the lack of effective host resistance, management of this disease requires frequent fungicide applications, which greatly increase the economic and environmental costs to produce banana. Weekly applications in most banana plantations lead to rapid evolution of fungicide-resistant strains within populations causing disease-control failures throughout the world. Given its extremely high economic importance, two strains of P. fijiensis were sequenced and assembled with the aid of a new genetic linkage map. The 74-Mb genome of P. fijiensis is massively expanded by LTR retrotransposons, making it the largest genome within the Dothideomycetes. Melting-curve assays suggest that the genomes of two closely related members of the Sigatoka disease complex, P. eumusae and P. musae, also are expanded. Electrophoretic karyotyping and analyses of molecular markers in P. fijiensis field populations showed chromosome-length polymorphisms and high genetic diversity. Genetic differentiation was also detected using neutral markers, suggesting strong selection with limited gene flow at the studied geographic scale. Frequencies of fungicide resistance in fungicide-treated plantations were much higher than those in untreated wild-type P. fijiensis populations. A homologue of the Cladosporium fulvum Avr4 effector, PfAvr4, was identified in the P. fijiensis genome. Infiltration of the purified PfAVR4 protein into leaves of the resistant banana variety Calcutta 4 resulted in a hypersensitive-like response. This result suggests that Calcutta 4 could carry an unknown resistance gene recognizing PfAVR4. Besides adding to our understanding of the overall Dothideomycete genome structures, the P. fijiensis genome will aid in developing fungicide treatment schedules to combat this pathogen and in improving the efficiency of banana breeding programs.
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http://dx.doi.org/10.1371/journal.pgen.1005876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4981457PMC
August 2016

The battle in the apoplast: further insights into the roles of proteases and their inhibitors in plant-pathogen interactions.

Front Plant Sci 2015 3;6:584. Epub 2015 Aug 3.

Laboratory of Phytopathology, Wageningen University and Research Centre , Wageningen, Netherlands.

Upon host penetration, fungal pathogens secrete a plethora of effectors to promote disease, including proteases that degrade plant antimicrobial proteins, and protease inhibitors (PIs) that inhibit plant proteases with antimicrobial activity. Conversely, plants secrete proteases and PIs to protect themselves against pathogens or to mediate recognition of pathogen proteases and PIs, which leads to induction of defense responses. Many examples of proteases and PIs mediating effector-triggered immunity in host plants have been reported in the literature, but little is known about their role in compromising basal defense responses induced by microbe-associated molecular patterns. Recently, several reports appeared in literature on secreted fungal proteases that modify or degrade pathogenesis-related proteins, including plant chitinases or PIs that compromise their activities. This prompted us to review the recent advances on proteases and PIs involved in fungal virulence and plant defense. Proteases and PIs from plants and their fungal pathogens play an important role in the arms race between plants and pathogens, which has resulted in co-evolutionary diversification and adaptation shaping pathogen lifestyles.
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http://dx.doi.org/10.3389/fpls.2015.00584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4522555PMC
August 2015

Flexible gateway constructs for functional analyses of genes in plant pathogenic fungi.

Fungal Genet Biol 2015 Jun;79:186-92

Laboratory of Phytopathology, Wageningen University, Wageningen, The Netherlands.

Genetic manipulation of fungi requires quick, low-cost, efficient, high-throughput and molecular tools. In this paper, we report 22 entry constructs as new molecular tools based on the Gateway technology facilitating rapid construction of binary vectors that can be used for functional analysis of genes in fungi. The entry vectors for single, double or triple gene-deletion mutants were developed using hygromycin, geneticin and nourseothricin resistance genes as selection markers. Furthermore, entry vectors containing green fluorescent (GFP) or red fluorescent (RFP) in combination with hygromycin, geneticin or nourseothricin selection markers were generated. The latter vectors provide the possibility of gene deletion and simultaneous labelling of the fungal transformants with GFP or RFP reporter genes. The applicability of a number of entry vectors was validated in Zymoseptoria tritici, an important fungal wheat pathogen.
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http://dx.doi.org/10.1016/j.fgb.2015.03.016DOI Listing
June 2015

FPLC and liquid-chromatography mass spectrometry identify candidate necrosis-inducing proteins from culture filtrates of the fungal wheat pathogen Zymoseptoria tritici.

Fungal Genet Biol 2015 Jun;79:54-62

Wageningen University & Research Center, Plant Research International, 6708 PB Wageningen, The Netherlands. Electronic address:

Culture filtrates (CFs) of the fungal wheat pathogen Zymoseptoria tritici were assayed for necrosis-inducing activity after infiltration in leaves of various wheat cultivars. Active fractions were partially purified and characterized. The necrosis-inducing factors in CFs are proteinaceous, heat stable and their necrosis-inducing activity is temperature and light dependent. The in planta activity of CFs was tested by a time series of proteinase K (PK) co-infiltrations, which was unable to affect activity 30min after CF infiltrations. This suggests that the necrosis inducing proteins (NIPs) are either absent from the apoplast and likely actively transported into mesophyll cells or protected from the protease by association with a receptor. Alternatively, plant cell death signaling pathways might be fully engaged during the first 30min and cannot be reversed even after PK treatment. Further fractionation of the CFs with the highest necrosis-inducing activity involved fast performance liquid chromatography, SDS-PAGE and mass spectrometry. This revealed that most of the proteins present in the fractions have not been described before. The two most prominent ZtNIP encoding candidates were heterologously expressed in Pichia pastoris and subsequent infiltration assays showed their differential activity in a range of wheat cultivars.
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http://dx.doi.org/10.1016/j.fgb.2015.03.015DOI Listing
June 2015

Proteome catalog of Zymoseptoria tritici captured during pathogenesis in wheat.

Fungal Genet Biol 2015 Jun;79:42-53

Wageningen University & Research Center, Plant Research International, 6708PB Wageningen, The Netherlands. Electronic address:

Zymoseptoria tritici is an economically important pathogen of wheat. However, the molecular basis of pathogenicity on wheat is still poorly understood. Here, we present a global survey of the proteins secreted by this fungus in the apoplast of resistant (cv. Shafir) and susceptible (cv. Obelisk) wheat cultivars after inoculation with reference Z. tritici strain IPO323. The fungal proteins present in apoplastic fluids were analyzed by gel electrophoresis and by data-independent acquisition liquid chromatography/mass spectrometry (LC/MS(E)) combined with data-dependent acquisition LC-MS/MS. Subsequent mapping mass spectrometry-derived peptide sequence data against the genome sequence of strain IPO323 identified 665 peptides in the MS(E) and 93 in the LC-MS/MS mode that matched to 85 proteins. The identified fungal proteins, including cell-wall degrading enzymes and proteases, might function in pathogenicity, but the functions of many remain unknown. Most fungal proteins accumulated in cv. Obelisk at the onset of necrotrophy. This inventory provides an excellent basis for future detailed studies on the role of these genes and their encoded proteins during pathogenesis in wheat.
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http://dx.doi.org/10.1016/j.fgb.2015.04.018DOI Listing
June 2015

Monitoring Three Resistance Genes in Iranian Sunflower Inbred Lines.

Iran J Biotechnol 2015 Jun;13(2):45-50

Seed and Plant Improvement Institute (SPII), Karaj, Iran.

Background: Downy mildew caused by is a devastating disease in sunflower worldwide. Several dominant resistance genes designated as have been identified and linked molecular markers have been demonstrated. However, no information on theresistance genes is available forIranian lines.

Objectives: The presence of three map-based molecular markers previously proved to be linked to different resistance genes were evaluated in sunflower inbred lines.

Materials And Methods: Using PCR-based and CAPS molecular markers, 26 sunflower inbred lines with different responses to race 100 were used to detect the presence of three resistance loci including , and within the lines.

Results: Molecular marker linked to was present in some of the sunflower lines but was not correlated with the phenotypic reaction of the lines to race 100. Despite the use of three markers linked to , PCR failed to amplify any corresponding product. This data may suggest that none of the genotypes possessed locus. -linked cleaved amplified polymorphic sequences (CAPS) were present in several resistance lines and effectively differentiated susceptible and resistant sunflower lines.

Conclusions: Applicability of molecular markers in breeding programs revisited in disease management.
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http://dx.doi.org/10.15171/ijb.1047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5435005PMC
June 2015

Genetic Diversity of Isolates, the Causal Agent of Wheat Tan Spot Disease from Northern Iran.

Iran J Biotechnol 2015 Jun;13(2):39-44

Department of Agronomy and Plant Breeding, Sanandaj Branch, Islamic Azad University, Sanandaj, Iran.

Background: The tan spot disease of wheat caused by has become a major disease in most wheat growing areas worldwide.

Objectives: Here we used ISSR and RAPD markers to study the genetic diversity of 34 isolates collected from North of Iran.

Materials And Methods: The leaves having the typical symptoms of tan spot disease were collected and after fungal isolation, purification and identification, DNAs were extracted. After PCR amplification using each primer, PCR products were run in agarose gels, and the resulting bands were scored. Cluster analysis was performed using Un-Wighted Nighbor Joining method.

Results: A total of 178 reproducible bands were scored. Out of which 115 (65%) were polymorphic corresponding to an average of 8 polymorphic bands per primer. The average PIC values for ISSR and RAPD markers were 0.38 and 0.43, respectively. A high degree of genetic variability among Iranian isolates was identified. Cluster analysis based on un-weighted neighbor-joining method using the combined molecular data revealed five distinct clusters. The results from the cluster analysis indicated that the genetic similarity among the Iranian isolates could be partly explained by geographic origins where the isolates were collected.

Conclusions: Genetic variability of along with relatively high level of geographic diversity observed in this study may suggest longer evolutionary period for the isolates from the Middle East, wheat center of origin, as opposed to other places.
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http://dx.doi.org/10.15171/ijb.1118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5435004PMC
June 2015

Synergistic Action of a Metalloprotease and a Serine Protease from Fusarium oxysporum f. sp. lycopersici Cleaves Chitin-Binding Tomato Chitinases, Reduces Their Antifungal Activity, and Enhances Fungal Virulence.

Mol Plant Microbe Interact 2015 Sep 10;28(9):996-1008. Epub 2015 Aug 10.

1 Laboratory of Phytopathology, Wageningen University and Research Centre, 6708 PB, Wageningen, The Netherlands;

As part of their defense strategy against fungal pathogens, plants secrete chitinases that degrade chitin, the major structural component of fungal cell walls. Some fungi are not sensitive to plant chitinases because they secrete chitin-binding effector proteins that protect their cell wall against these enzymes. However, it is not known how fungal pathogens that lack chitin-binding effectors overcome this plant defense barrier. Here, we investigated the ability of fungal tomato pathogens to cleave chitin-binding domain (CBD)-containing chitinases and its effect on fungal virulence. Four tomato CBD chitinases were produced in Pichia pastoris and were incubated with secreted proteins isolated from seven fungal tomato pathogens. Of these, Fusarium oxysporum f. sp. lycopersici, Verticillium dahliae, and Botrytis cinerea were able to cleave the extracellular tomato chitinases SlChi1 and SlChi13. Cleavage by F. oxysporum removed the CBD from the N-terminus, shown by mass spectrometry, and significantly reduced the chitinase and antifungal activity of both chitinases. Both secreted metalloprotease FoMep1 and serine protease FoSep1 were responsible for this cleavage. Double deletion mutants of FoMep1 and FoSep1 of F. oxysporum lacked chitinase cleavage activity on SlChi1 and SlChi13 and showed reduced virulence on tomato. These results demonstrate the importance of plant chitinase cleavage in fungal virulence.
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http://dx.doi.org/10.1094/MPMI-04-15-0074-RDOI Listing
September 2015

Novel Mutations Detected in Avirulence Genes Overcoming Tomato Cf Resistance Genes in Isolates of a Japanese Population of Cladosporium fulvum.

PLoS One 2015 22;10(4):e0123271. Epub 2015 Apr 22.

Wageningen University, Laboratory of Phytopathology, Wageningen, The Netherlands; Centre for Biosystems Genomics, Wageningen, The Netherlands.

Leaf mold of tomato is caused by the biotrophic fungus Cladosporium fulvum which complies with the gene-for-gene system. The disease was first reported in Japan in the 1920s and has since been frequently observed. Initially only race 0 isolates were reported, but since the consecutive introduction of resistance genes Cf-2, Cf-4, Cf-5 and Cf-9 new races have evolved. Here we first determined the virulence spectrum of 133 C. fulvum isolates collected from 22 prefectures in Japan, and subsequently sequenced the avirulence (Avr) genes Avr2, Avr4, Avr4E, Avr5 and Avr9 to determine the molecular basis of overcoming Cf genes. Twelve races of C. fulvum with a different virulence spectrum were identified, of which races 9, 2.9, 4.9, 4.5.9 and 4.9.11 occur only in Japan. The Avr genes in many of these races contain unique mutations not observed in races identified elsewhere in the world including (i) frameshift mutations and (ii) transposon insertions in Avr2, (iii) point mutations in Avr4 and Avr4E, and (iv) deletions of Avr4E, Avr5 and Avr9. New races have developed by selection pressure imposed by consecutive introductions of Cf-2, Cf-4, Cf-5 and Cf-9 genes in commercially grown tomato cultivars. Our study shows that molecular variations to adapt to different Cf genes in an isolated C. fulvum population in Japan are novel but overall follow similar patterns as those observed in populations from other parts of the world. Implications for breeding of more durable C. fulvum resistant varieties are discussed.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0123271PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4406682PMC
January 2016

Role of electron transport chain of chloroplasts in oxidative burst of interaction between Erwinia amylovora and host cells.

Photosynth Res 2015 May 28;124(2):231-42. Epub 2015 Mar 28.

Horticulture Research Department, Seed and Plant Improvement Institute, Karaj, Iran,

Erwinia amylovora is a necrogenic bacterium, causing the fire blight disease on many rosaceous plants. Triggering oxidative burst by E. amylovora is a key response by which host plants try to restrain pathogen spread. Electron transport chain (ETC) of chloroplasts is known as an inducible source of reactive oxygen species generation in various stresses. This research was performed to assess the role of this ETC in E. amylovora-host interaction using several inhibitors of this chain in susceptible and resistant apple and pear genotypes. All ETC inhibitors delayed appearance of disease necrosis, but the effects of methyl viologen, glutaraldehyde, and DCMU were more significant. In the absence of inhibitors, resistant genotypes showed an earlier and severe H2O2 generation and early suppression of redox dependent, psbA gene. The effects of inhibitors were corresponding to the redox potential of ETC inhibitory sites. In addition, delayed necrosis appearance was associated with the decreased disease severity and delayed H2O2 generation. These results provide evidences for the involvement of this ETC in host oxidative burst and suggest that chloroplast ETC has significant role in E. amylovora-host interaction.
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http://dx.doi.org/10.1007/s11120-015-0127-8DOI Listing
May 2015

Effector discovery in the fungal wheat pathogen Zymoseptoria tritici.

Mol Plant Pathol 2015 Dec 28;16(9):931-45. Epub 2015 Apr 28.

Wageningen University and Research Centre, Plant Research International, Droevendaalsesteeg 1, 6708, PB, Wageningen, The Netherlands.

Fungal plant pathogens, such as Zymoseptoria tritici (formerly known as Mycosphaerella graminicola), secrete repertoires of effectors to facilitate infection or trigger host defence mechanisms. The discovery and functional characterization of effectors provides valuable knowledge that can contribute to the design of new and effective disease management strategies. Here, we combined bioinformatics approaches with expression profiling during pathogenesis to identify candidate effectors of Z. tritici. In addition, a genetic approach was conducted to map quantitative trait loci (QTLs) carrying putative effectors, enabling the validation of both complementary strategies for effector discovery. In planta expression profiling revealed that candidate effectors were up-regulated in successive waves corresponding to consecutive stages of pathogenesis, contrary to candidates identified by QTL mapping that were, overall, expressed at low levels. Functional analyses of two top candidate effectors (SSP15 and SSP18) showed their dispensability for Z. tritici pathogenesis. These analyses reveal that generally adopted criteria, such as protein size, cysteine residues and expression during pathogenesis, may preclude an unbiased effector discovery. Indeed, genetic mapping of genomic regions involved in specificity render alternative effector candidates that do not match the aforementioned criteria, but should nevertheless be considered as promising new leads for effectors that are crucial for the Z. tritici-wheat pathosystem.
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http://dx.doi.org/10.1111/mpp.12251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6638447PMC
December 2015

The effect of drought stress on the expression of key genes involved in the biosynthesis of triterpenoid saponins in liquorice (Glycyrrhiza glabra).

Phytochemistry 2014 Jul 23;103:32-37. Epub 2014 Apr 23.

Cereal Research Dept., Seed and Plant Improvement Institute, Karaj, Islamic Republic of Iran.

Glycyrrhiza glabra is an important medicinal plant throughout the world. Glycyrrhizin is a triterpenoid that is among the most important secondary metabolites produced by liquorice. Drought stress is proposed to enhance the levels of secondary metabolites. In this study, the effect of drought stress on the expression of important genes involved in the glycyrrhizin biosynthetic pathway was examined. Drought stress at the seedling stage was applied to 8-day-old plants using polyethylene glycol. Subsequently, the samples were collected 0, 4, 8 or 24 h post-treatment. At the adult plant stage, 10-month-old plants were subjected to drought stress by discontinuing irrigation. Subsequently, samples were collected at 2, 16 and 28 days after drought imposition (S(2d), S(16d) and S(28d), respectively). We performed semi-quantitative RT-PCR assays to evaluate the gene expression levels of sequalene synthase (SQS), β-amyrin synthase (bAS), lupeol synthase (LUS) and cycloartenol synthase (CAS) during stress. Finally, the glycyrrhizin content of stolons was determined via HPLC. The results revealed that due to osmotic stress, the gene expression levels of SQS and bAS were increased, whereas those of CAS were relatively unchanged at the seedling stage. At the adult plant stage, the expression levels of SQS and bAS were increased under drought stress conditions, whereas the gene expression level of CAS remained relatively constant. The glycyrrhizin content in stolons was increased only under severe drought stress conditions (S(28d)). Our results indicate that application of controlled drought stress up-regulates the expression of key genes involved in the biosynthesis of triterpenoid saponins and directly enhances the production of secondary metabolites, including glycyrrhizin, in liquorice plants.
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http://dx.doi.org/10.1016/j.phytochem.2014.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7111703PMC
July 2014

Molecular characterization and functional analyses of ZtWor1, a transcriptional regulator of the fungal wheat pathogen Zymoseptoria tritici.

Mol Plant Pathol 2014 May 17;15(4):394-405. Epub 2013 Dec 17.

Plant Science Group, Plant Research International BV, Wageningen University, Droevendaalsesteeg 1, 06708 PB, Wageningen, The Netherlands; Department of Plant Protection, College of Agriculture, University of Tehran, Plant Pathology Building, Karaj, Iran.

Zymoseptoria tritici causes the major fungal wheat disease septoria tritici blotch, and is increasingly being used as a model for transmission and population genetics, as well as host-pathogen interactions. Here, we study the biological function of ZtWor1, the orthologue of Wor1 in the fungal human pathogen Candida albicans, as a representative of a superfamily of regulatory proteins involved in dimorphic switching. In Z. tritici, this gene is pivotal for pathogenesis, as ZtWor1 mutants were nonpathogenic and complementation restored the wild-type phenotypes. In planta expression analyses showed that ZtWor1 is up-regulated during the initiation of colonization and fructification, and regulates candidate effector genes, including one that was discovered after comparative proteome analysis of the Z. tritici wild-type strain and the ZtWor1 mutant, which was particularly expressed in planta. Cell fusion and anastomosis occur frequently in ZtWor1 mutants, reminiscent of mutants of MgGpb1, the β-subunit of the heterotrimeric G protein. Comparative expression of ZtWor1 in knock-out strains of MgGpb1 and MgTpk2, the catalytic subunit of protein kinase A, suggests that ZtWor1 is downstream of the cyclic adenosine monophosphate (cAMP) pathway that is crucial for pathogenesis in many fungal plant pathogens.
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http://dx.doi.org/10.1111/mpp.12102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6638687PMC
May 2014

The genomes of the fungal plant pathogens Cladosporium fulvum and Dothistroma septosporum reveal adaptation to different hosts and lifestyles but also signatures of common ancestry.

PLoS Genet 2012 29;8(11):e1003088. Epub 2012 Nov 29.

Laboratory of Phytopathology, Wageningen University, Wageningen, The Netherlands.

We sequenced and compared the genomes of the Dothideomycete fungal plant pathogens Cladosporium fulvum (Cfu) (syn. Passalora fulva) and Dothistroma septosporum (Dse) that are closely related phylogenetically, but have different lifestyles and hosts. Although both fungi grow extracellularly in close contact with host mesophyll cells, Cfu is a biotroph infecting tomato, while Dse is a hemibiotroph infecting pine. The genomes of these fungi have a similar set of genes (70% of gene content in both genomes are homologs), but differ significantly in size (Cfu >61.1-Mb; Dse 31.2-Mb), which is mainly due to the difference in repeat content (47.2% in Cfu versus 3.2% in Dse). Recent adaptation to different lifestyles and hosts is suggested by diverged sets of genes. Cfu contains an α-tomatinase gene that we predict might be required for detoxification of tomatine, while this gene is absent in Dse. Many genes encoding secreted proteins are unique to each species and the repeat-rich areas in Cfu are enriched for these species-specific genes. In contrast, conserved genes suggest common host ancestry. Homologs of Cfu effector genes, including Ecp2 and Avr4, are present in Dse and induce a Cf-Ecp2- and Cf-4-mediated hypersensitive response, respectively. Strikingly, genes involved in production of the toxin dothistromin, a likely virulence factor for Dse, are conserved in Cfu, but their expression differs markedly with essentially no expression by Cfu in planta. Likewise, Cfu has a carbohydrate-degrading enzyme catalog that is more similar to that of necrotrophs or hemibiotrophs and a larger pectinolytic gene arsenal than Dse, but many of these genes are not expressed in planta or are pseudogenized. Overall, comparison of their genomes suggests that these closely related plant pathogens had a common ancestral host but since adapted to different hosts and lifestyles by a combination of differentiated gene content, pseudogenization, and gene regulation.
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http://dx.doi.org/10.1371/journal.pgen.1003088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3510045PMC
May 2013

Phytotoxic secondary metabolites and peptides produced by plant pathogenic Dothideomycete fungi.

FEMS Microbiol Rev 2013 Jan 29;37(1):67-93. Epub 2012 Aug 29.

Department of Plant Pathology, University of California Davis, Davis, CA, USA.

Many necrotrophic plant pathogenic fungi belonging to the class of Dothideomycetes produce phytotoxic metabolites and peptides that are usually required for pathogenicity. Phytotoxins that affect a broad range of plant species are known as non-host-specific toxins (non-HSTs), whereas HSTs affect only a particular plant species or more often genotypes of that species. For pathogens producing HSTs, pathogenicity and host specificity are largely defined by the ability to produce the toxin, while plant susceptibility is dependent on the presence of the toxin target. Non-HSTs are not the main determinants of pathogenicity but contribute to virulence of the producing pathogen. Dothideomycetes are remarkable for the production of toxins, particularly HSTs because they are the only fungal species known so far to produce them. The synthesis, regulation, and mechanisms of action of the most important HSTs and non-HSTs will be discussed. Studies on the mode of action of HSTs have highlighted the induction of programed cell death (PCD) as an important mechanism. We discuss HST-induced PCD and the plant hypersensitive response upon recognition of avirulence factors that share common pathways. In this respect, although nucleotide-binding-site-leucine-rich repeat types of resistance proteins mediate resistance against biotrophs, they can also contribute to susceptibility toward necrotrophs.
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http://dx.doi.org/10.1111/j.1574-6976.2012.00349.xDOI Listing
January 2013

Karyotyping methods for fungi.

Methods Mol Biol 2012 ;835:591-602

Seed and Plant Improvement Institute, Karaj, Iran.

Pulsed field gel electrophoresis enables separation of fungal chromosomes up to several megabases and is a worthwhile tool for fungal karyotyping. The germ tube burst method is a technique to separate fungal chromosomes of any size for chromosome number determination as well as in situ hybridization. Here we provide detailed protocols for both complementary methods that have many applications in fungal biology including chromosome size and chromosome number polymorphisms, and in situ localization of genes on chromosomes.
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http://dx.doi.org/10.1007/978-1-61779-501-5_37DOI Listing
April 2012

MAP kinase phosphorylation and cAMP assessment in fungi.

Methods Mol Biol 2012 ;835:571-83

Seed and Plant Improvement Institute, Karaj, Iran.

The cyclic AMP (cAMP) signaling and mitogen-activated protein (MAP) kinase pathways are the most important signal transduction pathways in eukaryotes. In many plant pathogenic fungi they play pivotal roles in virulence and development. Identification and understanding the role of signal transduction pathways in regulation of cellular responses require robust biochemical techniques. Determination of both the phosphorylation status of MAPKs and the intracellular levels of cAMP is required to unravel the function of these pathways during adaptation of fungi to environmental stress conditions or when particular fungal genes are disrupted or silenced. Here we describe protocols to determine the phosphorylation status of three different MAPKs including Fus3, Slt2 and Hog1 as well as a protocol to measure the intracellular levels of cAMP levels. These protocols can be adapted for a wide range of fungi.
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http://dx.doi.org/10.1007/978-1-61779-501-5_35DOI Listing
April 2012

Finished genome of the fungal wheat pathogen Mycosphaerella graminicola reveals dispensome structure, chromosome plasticity, and stealth pathogenesis.

PLoS Genet 2011 Jun 9;7(6):e1002070. Epub 2011 Jun 9.

USDA-Agricultural Research Service, Purdue University, West Lafayette, Indiana, United States of America.

The plant-pathogenic fungus Mycosphaerella graminicola (asexual stage: Septoria tritici) causes septoria tritici blotch, a disease that greatly reduces the yield and quality of wheat. This disease is economically important in most wheat-growing areas worldwide and threatens global food production. Control of the disease has been hampered by a limited understanding of the genetic and biochemical bases of pathogenicity, including mechanisms of infection and of resistance in the host. Unlike most other plant pathogens, M. graminicola has a long latent period during which it evades host defenses. Although this type of stealth pathogenicity occurs commonly in Mycosphaerella and other Dothideomycetes, the largest class of plant-pathogenic fungi, its genetic basis is not known. To address this problem, the genome of M. graminicola was sequenced completely. The finished genome contains 21 chromosomes, eight of which could be lost with no visible effect on the fungus and thus are dispensable. This eight-chromosome dispensome is dynamic in field and progeny isolates, is different from the core genome in gene and repeat content, and appears to have originated by ancient horizontal transfer from an unknown donor. Synteny plots of the M. graminicola chromosomes versus those of the only other sequenced Dothideomycete, Stagonospora nodorum, revealed conservation of gene content but not order or orientation, suggesting a high rate of intra-chromosomal rearrangement in one or both species. This observed "mesosynteny" is very different from synteny seen between other organisms. A surprising feature of the M. graminicola genome compared to other sequenced plant pathogens was that it contained very few genes for enzymes that break down plant cell walls, which was more similar to endophytes than to pathogens. The stealth pathogenesis of M. graminicola probably involves degradation of proteins rather than carbohydrates to evade host defenses during the biotrophic stage of infection and may have evolved from endophytic ancestors.
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http://dx.doi.org/10.1371/journal.pgen.1002070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3111534PMC
June 2011