Molecular modelling of the emergence of azole resistance in Mycosphaerella graminicola

A structural rationale for recent emergence of azole (imidazole and triazole) resistance associated with CYP51 mutations in the wheat pathogen Mycosphaerella graminicola is presented, attained by homology modelling of the wild type protein and 13 variant proteins. The novel molecular models of M. graminicola CYP51 are based on multiple homologues, individually identified for each variant, rather than using a single structural scaffold, providing a robust structure-function rationale for the binding of azoles, including important fungal specific regions for which no structural information is available. The wild type binding pocket reveals specific residues in close proximity to the bound azole molecules that are subject to alteration in the variants. This implicates azole ligands as important agents exerting selection on specific regions bordering the pocket, that become the focus of genetic mutation events, leading to reduced sensitivity to that group of related compounds. Collectively, the models account for several observed functional effects of specific alterations, including loss of triadimenol sensitivity in the Y137F variant, lower sensitivity to tebuconazole of I381V variants and increased resistance to prochloraz of V136A variants. Deletion of Y459 and G460, which brings about removal of that entire section of beta turn from the vicinity of the binding pocket, confers resistance to tebuconazole and epoxiconazole, but sensitivity to prochloraz in variants carrying a combination of A379G I381V ΔY459/G460. Measurements of binding pocket volume proved useful in assessment of scope for general resistance to azoles by virtue of their accommodation without bonding interaction, particularly when combined with analysis of change in positions of key amino acids. It is possible to predict the likely binding orientation of an azole molecule in any of the variant CYPs, providing potential for an in silico screening system and reliable predictive approach to assess the probability of particular variants exhibiting resistance to particular azole fungicides.     

Detection of strobilurin-resistant isolates of Mycosphaerella graminicola in Morocco

Septoria tritici blotch caused by Mycosphaerella graminicola (anamorph: Septoria tritici) is nowadays one of the most frequently occurring diseases on both bread and durum wheat crops. Two hundred and thirty isolates of the fungus were sampled from six distinct wheat-producing regions of Morocco in order to investigate the resistance of M. graminicola to strobilurins in this country, where this fungicide class is increasingly used in wheat-pest management. A subset of 134 isolates was first collected in 2008 from Meknes-Tafilalet, Tadla-Azilal, Gharb and Chaouia. Furthermore, 96 additional isolates were sampled in 2010 from the fourth regions investigated in 2008 plus Fes-Boulmane and Doukkala-Abda. Sensitivity or resistance within the isolates were determined by screening the G143A cytochrome b substitution conferring resistance. We used a mismatch amplification mutation assay allowing the amplification of either G143 (sensitive) or A143 (resistant) allele. All the 2008 isolates were found to be sensitive since they carry the wild-type allele G143. However, 9 (9%) out of the 2010 isolates were found to contain the resistant allele A143 and therefore to be resistant. Four of them were from Gharb and five from Fes-Boulmane. This study highlighted for the first time the occurrence of strobilurin-resistant isolates of M. graminicola in Morocco. Further genetic investigations should determine if the resistant isolates emerged independently in Morocco or traveled by wind-migration from Europe.     

Impact of recently emerged sterol 14{alpha}-demethylase (CYP51) variants of Mycosphaerella graminicola on azole fungicide sensitivity

The progressive decline in the effectiveness of some azole fungicides in controlling Mycosphaerella graminicola, causal agent of the damaging Septoria leaf blotch disease of wheat, has been correlated with the selection and spread in the pathogen population of specific mutations in the M. graminicola CYP51 (MgCYP51) gene encoding the azole target sterol 14α-demethylase. Recent studies have suggested that the emergence of novel MgCYP51 variants, often harboring substitution S524T, has contributed to a decrease in the efficacy of prothioconazole and epoxiconazole, the two currently most effective azole fungicides against M. graminicola. In this study, we establish which amino acid alterations in novel MgCYP51 variants have the greatest impact on azole sensitivity and protein function. We introduced individual and combinations of identified alterations by site-directed mutagenesis and functionally determined their impact on azole sensitivity by expression in a Saccharomyces cerevisiae mutant YUG37::erg11 carrying a regulatable promoter controlling native CYP51 expression. We demonstrate that substitution S524T confers decreased sensitivity to all azoles when introduced alone or in combination with Y461S. In addition, S524T restores the function in S. cerevisiae of MgCYP51 variants carrying the otherwise lethal alterations Y137F and V136A. Sensitivity tests of S. cerevisiae transformants expressing recently emerged MgCYP51 variants carrying combinations of alterations D134G, V136A, Y461S, and S524T reveal a substantial impact on sensitivity to the currently most widely used azoles, including epoxiconazole and prothioconazole. Finally, we exploit a recently developed model of the MgCYP51 protein to predict that the substantial structural changes caused by these novel combinations reduce azole interactions with critical residues in the binding cavity, thereby causing resistance.     

Mechanism of binding of prothioconazole to Mycosphaerella graminicola CYP51 differs from that of other azole antifungals

Prothioconazole is one of the most important commercially available demethylase inhibitors (DMIs) used to treat Mycosphaerella graminicola infection of wheat, but specific information regarding its mode of action is not available in the scientific literature. Treatment of wild-type M. graminicola (strain IPO323) with 5 μg of epoxiconazole, tebuconazole, triadimenol, or prothioconazole ml(-1) resulted in inhibition of M. graminicola CYP51 (MgCYP51), as evidenced by the accumulation of 14α-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol in azole-treated cells. Successful expression of MgCYP51 in Escherichia coli enabled us to conduct spectrophotometric assays using purified 62-kDa MgCYP51 protein. Antifungal-binding studies revealed that epoxiconazole, tebuconazole, and triadimenol all bound tightly to MgCYP51, producing strong type II difference spectra (peak at 423 to 429 nm and trough at 406 to 409 nm) indicative of the formation of classical low-spin sixth-ligand complexes. Interaction of prothioconazole with MgCYP51 exhibited a novel spectrum with a peak and trough observed at 410 nm and 428 nm, respectively, indicating a different mechanism of inhibition. Prothioconazole bound to MgCYP51 with 840-fold less affinity than epoxiconazole and, unlike epoxiconazole, tebuconazole, and triadimenol, which are noncompetitive inhibitors, prothioconazole was found to be a competitive inhibitor of substrate binding. This represents the first study to validate the effect of prothioconazole on the sterol composition of M. graminicola and the first on the successful heterologous expression of active MgCYP51 protein. The binding affinity studies documented here provide novel insights into the interaction of MgCYP51 with DMIs, especially for the new triazolinethione derivative prothioconazole.     

Epitomics: global profiling of immune response to disease using protein microarrays

The immune system retains memory of current and past infections and can sense the presence of cancer by elaborating autoantibodies to tumor proteins. In the presence of an autoimmune disease, the immune system is an efficient, natural biosensor. Therefore we exploit the immune system through a high-throughput process to isolate disease-specific epitopes for diagnostic and therapeutic purposes. These cloned disease-specific antigens are robotically spotted onto protein microarrays and interrogated with serum from the subjects under analyses. These arrays deliver personalized profiles of antigenic exposures and therapeutic targets for personalized immunotherapy. The immune system is the ultimate biosensor, superior to anything a human could create and ready to be exploited for biotechnology and biomedicine.     

Expression profiling in pancreas during the acute phase of pancreatitis using cDNA microarrays

Most attacks of acute pancreatitis are self-limiting, suggesting that the pancreatic cells adapt their phenotype to prevent progression of the disease. Such phenotypic change must involve a coordinated modification in the expression of numerous genes. To identify differentially expressed genes, high-density mouse cDNA microarrays were hybridized with cDNA probes from both healthy pancreas and pancreas affected by acute pancreatitis. From the 7981 mouse genes analyzed, 239 showed significant changes in their expression during the acute phase of pancreatitis. Among them, 107 genes were up-regulated whereas 132 were down-regulated. They include genes whose function was not previously related to pancreatitis, suggesting that they are involved in some way into the acute pancreatic response. Finally, 40% of differentially expressed genes corresponded to ESTs. Demonstration that a large quantity of unexpected or yet uncharacterized genes showed altered expression during acute pancreatitis underscores the interest of a genome-based investigation. Some of these genes are certainly involved in the cellular defense against pancreatitis and, as such, deserve being studied further.     

Transcription profiling of the early gravitropic response in Arabidopsis using high-density oligonucleotide probe microarrays

Studies of plant tropisms, the directed growth toward or away from external stimuli such as light and gravity, began more than a century ago. Yet biochemical, physiological, and especially molecular mechanisms of plant tropic responses remain for the most part unclear. We examined expression of 8,300 genes during early stages of the gravitropic response using high-density oligonucleotide probe microarrays. Approximately 1.7% of the genes represented on the array exhibited significant expression changes within the first 30 min of gravity stimulation. Among gravity-induced genes were a number of genes previously implicated to be involved in gravitropism. However, a much larger number of the identified genes have not been previously associated with gravitropism. Because reorientation of plants may also expose plants to mechanical perturbations, we also compared the effects of a gentle mechanical perturbation on mRNA levels during the gravity response. It was found that approximately 39% of apparently gravity-regulated genes were also regulated by the mechanical perturbation caused by plant reorientation. Our study revealed the induction of complex gene expression patterns as a consequence of gravitropic reorientation and points to an interplay between the gravitropic and mechanical responses and to the extreme sensitivity of plants to even very gentle mechanical perturbations.     

Distribution of airborne Mycosphaerella graminicola inoculum at the field scale

A network of 10 Burkard 7-day spore-recording traps was set up in the Walloon region in Belgium to monitor the airborne inoculum of wheat pathogens. Three spore traps were used to analyse the distribution of Mycosphaerella graminicola inoculum at the field scale, at 1 m above ground level. Two traps were set up in a wheat field 100 m apart. The third trap was placed 70 m away in a sugar beet field adjacent to the wheat field. Total DNA from each fragment of spore trap tape corresponding to 1 day sampling was extracted and the quantity of M. graminicola was assessed using real-time polymerase chain reaction (PCR) assay. The experiment was conducted from July to October 2009. Positive detections were obtained for between 33 and 36 days, depending on the spore traps. When detected, the daily quantities of cDNA, collected from a volume of 14.4 m3, fluctuated between 4.84E+00 and 6.10E+03. Correlation coefficients higher than 0,82 and no significant differences were observed between the quantities of M. graminicola collected by the three spore traps, indicating that, at 1 m above ground level, the distribution of inoculum can be considered as homogenous at the tested field scale. This study confirms that spore traps coupled with real-time PCR could be used to assess the airborne inoculum of M. graminicola and to understand the development of the disease at this scale.     

Migration patterns among global populations of the pathogenic fungus Mycosphaerella graminicola

DNA sequences from five nuclear loci and data from three microsatellites were collected from 360 isolates representing 14 globally distributed populations of the plant pathogenic fungus Mycosphaerella graminicola. Haplotype networks were constructed for the five sequence loci and population subdivision was assessed using Hudson's permutation test. Migration estimates were calculated using six regional populations for both the sequence and microsatellite loci. While subdivision was detected among the six regional populations, significant gene flow was indicated among some of the populations. The European and Israeli populations contributed the majority of historical immigrants to the New World. Migration estimates for microsatellite loci were used to infer more recent migration events among specific New World populations. We conclude that gene flow was an important factor in determining the demographic history of Mycosphaerella graminicola.     

Presymptomatic and quantitative detection of Mycosphaerella graminicola development in wheat using a real-time PCR assay

Presymptomatic and accurate diagnosis of Mycosphaerella graminicola leaf blotch is desirable for the disease prediction and the timely application of fungicides. To develop a sensitive PCR assay, four specific primer pairs were designed. They were more specific than three known specific primer pairs. Three of them could detect as little as 0.5 pg M. graminicola DNA in a conventional PCR. A real-time PCR assay was applied for monitoring the disease progression in both inoculated and naturally infected wheat plants using the primer pair ST-rRNA F/R. In inoculated plants, M. graminicola DNA could be detected immediately after inoculation and a steady increase was detected before visible symptoms appeared at 8 days. The rapid growth period took place between 6 and 16 days postinoculation. In the field, the disease progression in the top three leaf layers was followed during the epidemic period. The results were significantly correlated to the disease indices (R=0.8986) and also to the number of pycnidia per leaf (R=0.9227). These suggest that the real-time PCR assay is a reliable approach for the presymptomatic and accurate detection of M. graminicola development in the field.     

A group of expressed cDNA sequences from the wheat fungal leaf blotch pathogen, Mycosphaerella graminicola (Septoria tritici)

A group of expressed sequence tags (ESTs) from the wheat fungal pathogen Mycosphaerella graminicola utilizing ammonium as a nitrogen source has been analyzed. Single pass sequences of complementary DNAs from 986 clones were determined. Contig analysis and sequence comparisons allowed 704 unique ESTs (unigenes) to be identified, of which 148 appeared as multiple copies. Searches of the nrdb95 protein database at EMBL using the BLAST2x algorithm revealed 407 (57.8%) sequences that generated high to moderate high scoring pairs with proteins of known and unknown function. The rest of the sequences (297) showed either weak or no similarities to database entries. Among the unigenes with assigned function, 26.7% were involved in primary metabolism and 17.9% were associated with protein and RNA metabolism. Fewer clones were ascribed roles in signal transduction (4.9%), transport and secretion (6.1%), cell structure (3.1%), and cell division (3.6%). Approximately 18.1% of the identities found were to hypothetical or unknown proteins mainly from the yeasts Saccharomyces cerevisiae and Schizosaccaromyces pombe. Comparison of the 297 sequences with no clear function to other fungal ESTs in the public domain revealed 12 sequences that had high to moderate similarity to Neurospora crassa, Emericella (Aspergillus) nidulans, or Magnaporthe grisea sequences.