Project description:Ruminants are the most efficient herbivorous animals to transform plant biomass into edible products, principally thanks to the rumen microbiota that produces a large array of enzymes responsible for the hydrolysis of plant cell wall polysaccharides. Several biotic and abiotic factors influence the efficiency of fiber degradation, which can ultimately impact the animal productivity and health. To provide more insight on mechanisms involved in the modulation of fibrolytic activity, a functional DNA microarray targeting genes coding for key enzymes involved in cellulose and hemicellulose degradation by rumen microbiota was designed. The DNA microarray, designated FibroChip, was validated using targets of increasing complexity. This new tool demonstrated sensitivity and specificity as well as explorative and semi-quantitative potential. The FibroChip was designed with the objective to propose a high throughput tool that enables to get a rapid picture of the capacity of rumen microorganisms to degrade cellulose and hemicellulose based on a targeted metatranscriptomics approach. We chose to focus on a few number of genes by targeting main ruminal fibrolytic microorganisms and selected CAZyme families that may have a pivotal role in cellulose and hemicellulose degradation. The microarray targets the coding sequence of catalytic domains from 8 CAZyme families involved in cellulose and hemicellulose degradation (i.e. glycoside hydrolases GH5, GH9, GH10, GH11, GH43 and GH48, and carbohydrate esterases CE1 and CE6). Taken together, these families present complementary activities needed for the complete degradation of cellulose and hemicellulose. In total, 392 nucleic sequences encoding 363 GH and 29 CE were kept for the microarray design. These sequences originated from 41 bacterial species, 4 protozoal species and 10 fungal species. The microarray is composed of 1631 25-mer probes. GoArrays strategy consisting by associating 2 25-mer probes targeting the same gene (Rimour et al., 2005) was also emplyed to determine 2618 composite probes of 54-mers. Triplicate of probes of 25 and 54-mers were synthetized in situ on an Agilent 8x15K DNA microarray. The microarray contained also 382 Agilent internal control probes including positive controls, negative controls and quality control probes. Probes were randomly placed on the array to avoid position bias.

Project description:Agaricus bisporus is a soil-inhabiting fungus which is cultivated for production of white button mushrooms. A disease of A. bisporus has been previously described with a range of disease symptoms (yield loss, pinning delay, cap distortions and cap browning) which has been given collective name of “Mushroom Virus X” (MVX). The causes of this disease are not clear however prior to this research an association was found between the disease and double-stranded RNA molecules in the mushroom fruitbodies. The experiment was designed to examine causes and host responses of the disease causing the Brown Cap symptom in the cultivated mushroom A. bisporus. This microarray experiment was performed before the Agaricus bisporus genome was sequenced. The gene sequences used to design probes were from known and novel A. bisporus sequences and sequences of transcript fragments identified by Suppression Subtractive Hybridization of non-symptomatic and virus-diseased A. bisporus mushroom fruitbodies. The A. bisporus mushroom fruitbodies were grown on composted wheat straw using commercial cultivation procedures. The gene expression comparison was made of RNA isolated from 32 mushroom fruitbodies (Agaricus bisporus) samples: 20 samples from 5 separate virus-infected commercial mushroom farms with crops displaying the brown symptom (4 replicate samples per farm) and 12 samples from a non-infected crop grown at the University of Warwick. The precise composition of the viral load was the subject of this and future research/papers. Abstract of Manuscript submitted to Applied and Environmental Microbiology: Characterizing the viral agents causing brown cap mushroom disease of Agaricus bisporus by Daniel Eastwood, Julian Green, Helen Grogan, and Kerry Burton (Paper #AEM01093-15). The symptoms of viral infections of fungi range from cryptic to severe but there is little knowledge of the factors involved in this transition of fungal/viral interactions. Brown Cap Mushroom Disease of the cultivated Agaricus bisporus is economically important and represents a model system to describe this transition. Differentially expressed transcript fragments between mushrooms showing the symptoms of Brown Cap Mushroom Disease and control white non-infected mushrooms have been identified and sequenced. Ten of these RNA fragments have been found to be up-regulated over a thousand-fold between diseased and non-diseased tissue but are absent from the Agaricus bisporus genome sequence and hybridise to double-stranded RNA’s extracted from diseased tissue. We hypothesize these transcript fragments are viral and represent components of the disease-causing agent, a bipartite virus with similarities to the family Partitiviridae. The virus fragments were found at two distinct levels within infected mushrooms, at raised levels in infected, non-symptomatic, white coloured mushrooms and much greater levels (3,500-87,000 times greater) in infected mushrooms exhibiting brown colouration. In addition, differential screening revealed 9 up-regulated and 32 down-regulated host Agaricus bisporus transcripts. Chromametric analysis was able to distinguish colour differences between non-infected white mushrooms and white infected mushrooms at an early stage of mushroom growth. This method may be the basis for an ‘on-farm’ disease detection assay.

Project description:Agaricus bisporus is a soil-inhabiting fungus which is cultivated for production of white button mushrooms. A disease of A. bisporus has been previously described with a range of disease symptoms (yield loss, pinning delay, cap distortions and cap browning) which has been given collective name of “Mushroom Virus X” (MVX). The causes of this disease are not clear however prior to this research an association was found between the disease and double-stranded RNA molecules in the mushroom fruitbodies. The experiment was designed to examine causes and host responses of the disease causing the Brown Cap symptom in the cultivated mushroom A. bisporus. This microarray experiment was performed before the Agaricus bisporus genome was sequenced. The gene sequences used to design probes were from known and novel A. bisporus sequences and sequences of transcript fragments identified by Suppression Subtractive Hybridization of non-symptomatic and virus-diseased A. bisporus mushroom fruitbodies. The A. bisporus mushroom fruitbodies were grown on composted wheat straw using commercial cultivation procedures. The gene expression comparison was made of RNA isolated from 32 mushroom fruitbodies (Agaricus bisporus) samples: 20 samples from 5 separate virus-infected commercial mushroom farms with crops displaying the brown symptom (4 replicate samples per farm) and 12 samples from a non-infected crop grown at the University of Warwick. The precise composition of the viral load was the subject of this and future research/papers. Abstract of Manuscript submitted to Applied and Environmental Microbiology: Characterizing the viral agents causing brown cap mushroom disease of Agaricus bisporus by Daniel Eastwood, Julian Green, Helen Grogan, and Kerry Burton (Paper #AEM01093-15). The symptoms of viral infections of fungi range from cryptic to severe but there is little knowledge of the factors involved in this transition of fungal/viral interactions. Brown Cap Mushroom Disease of the cultivated Agaricus bisporus is economically important and represents a model system to describe this transition. Differentially expressed transcript fragments between mushrooms showing the symptoms of Brown Cap Mushroom Disease and control white non-infected mushrooms have been identified and sequenced. Ten of these RNA fragments have been found to be up-regulated over a thousand-fold between diseased and non-diseased tissue but are absent from the Agaricus bisporus genome sequence and hybridise to double-stranded RNA’s extracted from diseased tissue. We hypothesize these transcript fragments are viral and represent components of the disease-causing agent, a bipartite virus with similarities to the family Partitiviridae. The virus fragments were found at two distinct levels within infected mushrooms, at raised levels in infected, non-symptomatic, white coloured mushrooms and much greater levels (3,500-87,000 times greater) in infected mushrooms exhibiting brown colouration. In addition, differential screening revealed 9 up-regulated and 32 down-regulated host Agaricus bisporus transcripts. Chromametric analysis was able to distinguish colour differences between non-infected white mushrooms and white infected mushrooms at an early stage of mushroom growth. This method may be the basis for an ‘on-farm’ disease detection assay. A gene expression comparison was made between diseased mushroom displaying the brown cap symptom with characteristic double-strand RNA profiles (banding pattern on gels) and non-symptomatic virus-free mushrooms. In total RNA was isolated from 32 mushroom fruitbody (Agaricus bisporus) samples: 20 samples from 5 separate virus-infected commercial mushroom farms with crops displaying the brown symptom (4 replicate samples per farm) and 12 samples from a non-infected crop grown at the University of Warwick. Commercially-grown mushrooms are produced in “flushes” at approximately weekly intervals. The samples were collected from commercial farms when symptoms were reported to us but these were from different flushes: Farm1 from the 2nd flush; Farm 2 from the 1st flush; Farm 3 from the 3rd flush; Farm 4 from the 1st flush; and Farm 9 from the 1st flush. To allow for comparisons on the basis of Flush Number, the non-infected mushrooms grown at the University of Warwick were sampled from the first, second and third flushes, 4 mushrooms sampled from each flush.

			Action	DRS
	11_1.msf	Msf
	11_2.msf	Msf
	14_1.msf	Msf
	14_2.msf	Msf
	16_1.msf	Msf

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Uncovering the abilities of Agaricus bisporus to degrade plant biomass throughout its life cycle

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Uncovering the abilities of Agaricus bisporus to degrade plant biomass throughout its life cycle.

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