Monthly Archives: January 2012

Searching For Candidate Effector Proteins of Rust Fungi

The understanding of effector proteins has advanced by leaps and bounds in the last few years.  Secreted by microorganisms interacting with plants, these small proteins enter a host cell and modify physiological changes, most notably influencing the suppression or activation of host directed immunity.  Fungal effector proteins have been characterized in the pathogen infection process as well as the suppression of host defenses in mutualistic associations such as mycorrhizae.  There is a recently published book, edited by Francis Martin & Sophien Kamoun, which addresses the current state of knowledge on the biology of microbial effector proteins.

Published on January 6th in the journal PLOS One, the paper “Using Hierarchical Clustering of Secreted Protein Families to Classify and Rank Candidate Effectors of Rust Fungi” authored by Saunders et al., seeks to describe unknown effector proteins by exploring the diversity of secreted proteins of rust fungi.  The Kamoun Lab has been at the forefront of understanding effector biology in the fungi, and this paper is a significant contribution to understanding how rust fungi invoke pathogenicity on their host plants.

Rust fungi are a monophyletic group of pathogens which cause damage on many important economic crop plants.  In this study, the authors investigated two pathogens with sequenced genomes, Puccinia graminis f. sp. tritici, the cause of wheat stem rust, and poplar leaf rust, Melampsora larici-populina.  By developing an analysis pipeline, the authors inspected the secretome of both fungi to search for putative effector proteins and describe their structure and possible mode of infection into a plant.  Few plant defense mechanisms have been identified for rust fungi.  This study was considered a preliminary step to identify candidate rust effectors in the eventual selection of new resistance (R) genes in plant breeding programs and genome sequencing initiatives.

Eight families of putative effector proteins were identified in the secretomes of P. graminis f. sp. tritici and M. larici-populina, and a total of 6663 proteins were identified by the pipeline, with 2826 proteins containing secretion signal peptide regions.  Analysis of the protein motifs identified several conserved cysteine motifs common to other effector proteins previously characterized from fungal and oomycete plant pathogens.  Not surprisingly, the authors identified many previously unrecognized proteins with domains that exhibited similarity to known pathogenicity-related or haustorial-expressed fungal proteins.  Both P. graminis f. sp. tritici and M. larici-populina showed differences in the types of effectors secreted and the numbers of each putative effector tribe.

As I mentioned before, this study should be considered a first step in the identification of pathogenicity related effector proteins from rust fungi.  Next steps would include wet lab characterization and experimental validation for putative effectors identified in this paper.  Additional studies will be needed to address the functional expression of these proteins, as well as the R genes expressed in planta, during the infection process initiated by the rust fungus.  This paper provides an interesting priority list for further studies in this rapidly advancing area of understanding the biology of the intimacies of the plant-fungus interaction.

Common Toolbox Based On Necrotrophy In A Fungal Lineage

One of the most exciting aspects of the genomic revolution in biology is understanding the genetic mechanisms behind an organisms natural history.  Within the fungi it has been fascinating to begin to tease apart how seemingly similar life histories can be explained by disparate genomic landscapes, or opposingly, how completely different modes of survival can be explained by a common set of genes.  There has been a steady increase in research over the last few years documenting a large array of tools in the genomic toolbox.

A recently published paper in the journal PLOS One, authored by Andrew et al., entitled “Evidence for a Common Toolbox Based on Necrotrophy in a Fungal Lineage Spanning Necrotrophs, Biotrophs, Endophytes, Host Generalists and Specialists” comes from the Kohn Lab at the University of Toronto, which has long studied the fungal family Sclerotiniaceae.  The Sclerotiniaceae is a group of Ascomycetes known for being typically necrotic pathogens which are host generalists or specialists.

This is a great group of fungi to address questions like: why is one species necrotic while another is just biotrophic? …and why are some fungi specialists, while others generally infect plants without regard to taxonomy?  To address these questions the authors sampled across 52 strains of fungi in the Sclerotiniaceae representing 30 taxa covering the spectrum of host specificity/generality and trophic types.  They chose a suite of genes responsible for both general cell housekeeping (controls, etc.) and associated with pathogenicity and constructed phylogenies of these genes to observe relationships between these 52 strains.  Evidence of positive selection acting on these genes, as well as site-specific selection, was also assessed.  Lastly, the authors assessed the pathogenicity of these strains by initiating infection studies on Arabidopsis thaliana plants.

The authors found that there are at least two origins of biotrophy from a necrotrophic ancestor in the Sclerotiniaceae and that there is evidence of selection on all the genes associated with pathogenicity tested in this study.  The housekeeping genes in this study were used to control for the phylogenetic analysis and showed no evidence of positive selection as opposed to the pathogenicity genes.  Furthermore, likelihood analyses showed no statistical differences in the genes of strains from different trophic lifestyles, as well as host generalists and specialists.

Within this study on the Sclerotiniaceae, it appears that there is a common tool box of genes shared by the fungal strains studied here.  The level of expression of genes differed in this study, which could explain trophic and host specificity differences exhibited by these fungi.  It will be interesting when we have more genome sequences from this family to see how genome structure and rearrangements have contributed to the expression and diversity of genes associated with necrotrophy in this family of fungi.

Trying to Close ‘Open Access’

I’m sure you’re aware of the recently proposed (and three page!) Research Works Act (Bill H.R. 3699) that has been introduced in the House of Representatives here in the US?  If not, then you should read the bill, and then most especially read:

1. An Op-Ed piece ‘Research Bought, Then Paid For‘ in today’s New York Times from Michael Eisen.  See also this post from Michael Eisen’s Blog.

2. A post in the Scientific American Blogs called ‘Scientists, Fight For Access!‘ written by Kevin Zelnio (Twitter & Blog).

3.  A series of posts on the issue of public access to publicly funded research from Jonathan Eisen’s Blog, and see ‘Scientists Embrace Openness’ through the Journal Science.

2012 Workshop Series in Microbial Genomics & Metagenomics at JGI

There’s another series of workshops for both microbial genomics and metagenomics presented by the U.S. Department of Energy’s Joint Genome Institute in 2012.   These workshops include two days of seminars and three days of hands-on tutorials for both microbial genomics and metagenomics.  These workshops are centered on the use of the following bioinformatic tools: IMG, IMG/M, IMG-ER, IMG-EDU, VISTA, GREENGENES and ARB.  Registration for this workshop can be found here.

Human Pathogenic Types of the Fungus Fusarium Detected in Plumbing Drains

I recently wrote about a paper that surveyed the diversity of bacteria in public restrooms using metagenomic techniques.  While that paper focused on bacteria on bathroom surfaces, another recent paper – “Widespread Occurrence of Diverse Human Pathogenic Types of the Fungus Fusarium Detected in Plumbing Drains”, authored by Dylan Short and colleagues – focused specifically on probing the diversity of the large Ascomycete genus Fusarium found in sink drains, with specific focus on isolates that are human pathogens.

The authors sampled 471 drains – more than 95% of which were from public bathroom sinks – from 131 buildings throughout the mid-eastern to southern United States (and California too).  They selectively isolated Fusarium species from sink drains using cotton swabs and then streaked petri plates of Nash-Snyder Agar, which is a semi-selective medium containing the fungicide pentachloronitrobenzene.  The plates were inspected after the fungi had some time to grow, were propagated, and then verified as Fusarium species using microscopic morphology and DNA sequencing.

Six different loci – translation elongation factor (TEF), the internal transcribed spacer region (ITS) into the large ribosomal subunit (LSU), the nuclear rDNA intergenic spacer region (IGS), the RNA polymerase II large subunit (RPB2), portion of the alpha-tubulin (TUB) gene, and calmodulin (CAM) – were identified using Sanger sequencing to assess the diversity of Fusarium in the sink drains.  The sequence data was compared to an extensive database of the genus Fusarium maintained by the Geiser Lab and others.

Fusarium species were extremely common in sink drains; 66% of the sink samples – and 82% of all the buildings sampled – yielded at least one isolate.  These isolates could largely be placed within three Fusarium species complexes: the Fusarium solani species complex (62% of samples), the F. oxysporum species complex (28%), and the F. dimerum species complex (8.5%).  Sink drains from 91% of private residences and 80% of public buildings yielded Fusarium isolates.  Of all the buildings that yielded Fusarium within sink drains, approximately 80% contained one of the six major isolates recognized from human infections.

It is interesting to note that human infections from Fusarium species are rare, but the six most common Fusarium isolates found in sink drains are also the six most common involved in human infection.  The authors note that it’s apparent that people are in constant contact with these fungi within indoor environments.  It’s also notable that novel species complexes were identified using these techniques and that there was a wide phylogenetic breadth to the Fusarium isolates that were sampled from sink drains.

This paper is a substantial contribution to the growing literature documenting the indoor environment for fungi.  The next step would be to use metagenomic techniques – and marker loci for fungi to encompass a meta-taxonomic assessment – to identify all the fungi found in sink drains.