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Carbohydrate Active Enzyme Domains in Proteins

As a preliminary analysis to identify wood degrading enzymes and any potentially raised pathogenicity via increased ability to enter the tree proteins predicted in

1. Chalara fraxinea TGAC annotation 1
2. Postia placenta JGI annotation
and
3. Fusarium graminareum Broad annotation

were used in PFAMSCAN to identify proteins containing domains from the list below at rough cutoff E <=1e-6: (PFAM domains in carbohydrate active enzymes described in CAZY)

Counts of proteins containing each PFAM domain are shown in this figure (you may need to download to get the full version, its a big figure), Chalara proteins are represented on the outer track, Fusarium on the middle and Postia on the inner. Postia placenta is the brown rot fungus and is a capable digester of wood. The wheat pathogen Fusarium most often attacks the kernel. The carbohydrate enzyme protein domain in Chalara is much more similar to that in Postia than fusarium, indicating a potential for Chalara to be aggressively and actively entering the tree through the wood.

Both mating types of Hymenoscyphus pseudoalbidus (Chalara fraxinea) are present at nursery, recent planting and natural spread sites

The contributors

Gavin Hunter, Joan Webber, Stephen Hendry and Clive Brasier at Forest Research

Background

The ash dieback pathogen Hymenoscyphus pseudoalbidus is now to be considered established in eastern Britain (see distribution in this map) and samples regularly come into the laboratory at Forest Research for morphological and DNA-based molecular diagnosis. This has formed the nucleus of a culture collection of the pathogen which we are characterising, including analysis of mating type.

Analysis

We used the multiplex PCR method of Gross et al (2012) to identify the MAT 1-1 and MAT 1-2 idiomorphs or sexual mating types of H. pseudoalbidus. Sequences of the primer products were in all cases homologous to the MAT sequences reported by Gross et al (2012).
The mating types were determined for six isolates from individual trees at a single nursery in Lincolnshire; six isolates from individual trees at a single recent ash planting site in Leicestershire; and nine isolates from individual trees at seven different ‘natural spread’ or woodland sites in Suffolk and Norfolk.

Results

As shown in the Tables linked at the bottom, both mating types, MAT1-1 and MAT1-2, were present in each site type. The overall ratio of MAT1-1: MAT1-2types in the 21 isolates was 11 : 10.

Interpretation

  1. Ash nursery stock entering the UK can carry both mating types of H. pseudoalbidus onto a nursery site.
  2. Out-planting of ash nursery stock can also carry both mating types onto a new site.
  3. As already indicated by Diane Saunders (January 28) for Kenninghall Wood and Ashwellthorpe Wood (Norfolk), both mating types are present across ‘natural spread’ or woodland sites in eastern England.
  4. Across all samples the frequency of MAT1-1 and MAT1-2 types was close to 1:1. This is consistent with the previous report of MAT ratios of 1:1 in H. pseudoalbidus by Gross et al. (2012); and with what is expected for a regularly sexually outcrossing ascomycete.It is also consistent with reports of high genetic variability in H. pseudoalbidus elsewhere in Europe i.e. H. pseudoalbidus now being found in the UK is also likely to be regularly sexually outcrossing.

nornex_isolates_1

nornex_isolates_2

nornex_isolates_3

nornex_isolates_4

see also GitHub HP01 .. HP036

Is the high representation of fungal transcripts in the infected ash material unusual or alarming?

Contributors
B. Patrick Chapman and Mark Gijzen, Agriculture and Agri-Food Canada

To accelerate discovery on Chalara ash dieback disease, sequence data from the ‘interaction transcriptome’ has been made available for ‘crowdsource’ analysis. Two different samples of infected material, AT1 and AT2, have been released thus far. Previous posts describe the analysis of assembled transcripts and their likely origin, from the host plant (Fraxinus spp.) or fungus (Hymenoscyphus pseudoalbidus; syn. Chalara fraxinea) based upon best BLAST match. This work suggests that fungal transcripts may account for ~30% the AT1 and ~15% of the AT2 assembled transcripts (http://oadb.tsl.ac.uk/?p=196 and http://oadb.tsl.ac.uk/?p=284). A previous post suggested that the potentially high percentage of pathogen transcripts present in the AT1 sample is somewhat unusual for samples from infected plant tissues (How useful is the AT1 assembly? Posted 16 Dec http://oadb.tsl.ac.uk/?p=254).

Pathogen sequences can account for >50% of transcripts from diseased plant tissues
We do not believe that the high representation of fungal transcripts in the AT1 sample is necessarily unusual or alarming because past studies have shown that pathogen sequences can dominate interaction transcriptomes, especially during late infection stages. For example, 48 h after inoculation of soybean hypocotyls with the root rot pathogen Phytophthora sojae, 60 to 70% of the expressed sequence tags (ESTs) from the infected tissue were from P. sojae (Qutob et al. 2000). Likewise, 5 d after infection of Arabidopsis leaves with Hyaloperonospora arabidopsidis, 58% of non-redundant ESTs were determined to be of pathogen origin (Cabral et al. 2011). Analysis of large subunit ribosomal RNA (ls-rRNA) can also be used to estimate the fraction of host and pathogen RNA in mixed samples, in cases where the ls-rRNA is polymorphic in size for the two species. For the P. sojae and soybean interaction, this method showed that from 12 to 48 h after infection the proportion of pathogen RNA increased rapidly, from <10% (12 h) to 40% (24 h) and 70% (48 h) (Moy et al. 2004). For Arabidopsis leaves 5 d after infection with H. arabidopsidis, analysis of ls-rRNA showed that ~50% of RNA originated from the pathogen. Conclusion These examples demonstrate that the fraction of pathogen sequences in the interaction transcriptome may be large, at least for oomycete species such P. sojae and H. arabidopsidis infecting their natural hosts. Thus, we do not believe that it is particularly unusual or alarming that fungal sequences are estimated to account for 30% or more of the assembled transcripts in the AT1 sample. References Cabral A, Stassen JH, Seidl MF, Bautor J, Parker JE, Van den Ackerveken G (2011) Identification of Hyaloperonospora arabidopsidis transcript sequences expressed during infection reveals isolate-specific effectors. PLoS One 6:e19328 http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0019328 Moy P, Qutob D, Chapman BP, Atkinson I, Gijzen M (2004) Patterns of gene expression upon infection of soybean plants by Phytophthora sojae. Mol Plant Microbe Interact 17:1051-62 http://apsjournals.apsnet.org/doi/abs/10.1094/MPMI.2004.17.10.1051 Qutob D, Hraber PT, Sobral BW, Gijzen M (2000) Comparative analysis of expressed sequences in Phytophthora sojae. Plant Physiol 123:243-54 http://www.plantphysiol.org/content/123/1/243.long