RNA trafficking in plants contributes to local and long-distance coordination of plant development and response to the environment. However, investigations of mobile RNA identity and function are hindered by the inherent difficulty of tracing a given molecule of RNA from its cell of origin to its destination. Several methods have been used to address this problem, but all are limited to some extent by constraints associated with accurately sampling phloem sap or detecting trafficked RNA. Certain parasitic plant species form symplastic connections to their hosts and thereby provide an additional system for studying RNA trafficking. The haustorial connections of Cuscuta andPhelipanche species are similar to graft junctions in that they are able to transmit mRNAs, viral RNAs, siRNAs, and proteins from the host plants to the parasite. In contrast to other graft systems, these parasites form connections with host species that span a wide phylogenetic range, such that a high degree of nucleotide sequence divergence may exist between host and parasites and allow confident identification of most host RNAs in the parasite system. The ability to identify host RNAs in parasites, and vice versa, will facilitate genomics approaches to understanding RNA trafficking. This review discusses the nature of host–parasite connections and the potential significance of host RNAs for the parasite. Additional research on host–parasite interactions is needed to interpret results of RNA trafficking studies, but parasitic plants may provide a fascinating new perspective on RNA trafficking.
Gene transfer has been identified as a prevalent and pervasive phenomenon and an important source of genomic innovation in bacteria. The role of gene transfer in microbial eukaryotes seems to be of a reduced magnitude but in some cases can drive important evolutionary innovations, such as new functions that underpin the colonization of different niches. The aim of this review is to summarize published cases that support the hypothesis that horizontal gene transfer (HGT) has played a role in the evolution of phytopathogenic traits in fungi and oomycetes. Our survey of the literature identifies 46 proposed cases of transfer of genes that have a putative or experimentally demonstrable phytopathogenic function. When considering the life-cycle steps through which a pathogen must progress, the majority of the HGTs identified are associated with invading, degrading, and manipulating the host. Taken together, these data suggest HGT has played a role in shaping how fungi and oomycetes colonize plant hosts.
Rust fungi include many species that are devastating crop pathogens. To develop resistant plants, a better understanding of rust virulence factors, or effector proteins, is needed. Thus far, only six rust effector proteins have been described: AvrP123, AvrP4, AvrL567, AvrM, RTP1 and PGTAUSPE-10-1. Although some are well established model proteins used to investigate mechanisms of immune receptor activation (avirulence activities) or entry into plant cells, how they work inside host tissues to promote fungal growth remains unknown. The genome sequences of four rust fungi (two Melampsoraceae and two Pucciniaceae) have been analyzed so far. Genome-wide analyses of these species, as well as transcriptomics performed on a broader range of rust fungi, revealed hundreds of small secreted proteins considered as rust candidate secreted effector proteins (CSEPs). The rust community now needs high-throughput approaches (effectoromics) to accelerate effector discovery/characterization and to better understand how they function in planta. However, this task is challenging due to the non-amenability of rust pathosystems (obligate biotrophs infecting crop plants) to traditional molecular genetic approaches mainly due to difficulties in culturing these species in vitro. The use of heterologous approaches should be promoted in the future.
This paper is an excellent demonstration of the power of phylogenomics for the discovery of genes involved in traits of interest.
The authors report a larger scale genome comparison between symbiotic (arbuscular mycorrhiza forming) and non-symbiotic plant groups. They identify gene loss in plant species that go back to a minimum of four independent loss-of-symbiosis events; one in the Brassicales, one in the Caryophyllales (Amaranthaceae), one in the Laminales (Orobanchaceae) and one in the Fabales (Lupinus).
They performed an impressive phylogenomic analysis and identified a list of 300 Medicago genes that are present in most of the analyzed species but absent in all non-symbiotic Brassicaceae. Upon filtering the list further, by including paraphyletic non-symbiotic species, they arrived at a list of around 100 genes that were consistently absent in the non-mycorrhizal species. Lupinus as a plant that lost arbuscular mycorrhiza but maintained root nodule symbiosis was very informative because common symbiosis genes should be maintained in this genus.
The results are consistent with an evolutionary scenario in which each of the independent loss-of-symbiosis events, for which the loss of a single gene function was sufficient, was followed by a subsequent larger scale gene erosion that consistently removed the same orthologous genes in the four different clades.
This very interesting and largely unexpected observation reveals two opposing evolutionary forces that decide over the prevalence of this 'symbiosis-associated' gene set. On the one hand, the existing symbiosis leads to a successful maintenance of symbiosis genes. On the other hand, a yet unidentified force resulted in a consistent pattern of larger scale gene loss after each independent loss-of-symbiosis event. The forces behind this erosion must have acted either very quickly, before each of the non-symbiotic clades diversified from their respective common ancestor, or they independently led to consistent gene loss patterns after speciation.
Because symbiosis-related genes are overrepresented in the eroded gene set, it is likely that a large proportion, if not all of them, are of specific functional relevance in arbuscular mycorrhization (AM). Therefore this study is of major importance not only from an evolutionary perspective, but also because it demonstrates a novel strategy to identify candidate genes involved in AM symbiosis.
By Martin Parniske, F1000 Plant Biology, Biocenter University of Munich (LMU), Martinsried, Germany.
Disclosures - Martin Parniske has published a joint paper with the corresponding author in 2012.
The (1,3)-β-glucan callose is a major component of cell wall thickenings in response to pathogen attack in plants. GTPases have been suggested to regulate pathogen-induced callose biosynthesis. To elucidate the regulation of callose biosynthesis in Arabidopsis thaliana, we screened microarray data and identified transcriptional upregulation of the GTPase RabA4c after biotic stress. We studied the function of RabA4c in its native and dominant negative (dn) isoform inRabA4c overexpression lines. RabA4c overexpression caused complete penetration resistance to the virulent powdery mildew Golovinomyces cichoracearum due to enhanced callose deposition at early time points of infection, which prevented fungal ingress into epidermal cells. By contrast,RabA4c(dn) overexpression did not increase callose deposition or penetration resistance. A cross of the resistant line with the pmr4 disruption mutant lacking the stress-induced callose synthase PMR4 revealed that enhanced callose deposition and penetration resistance were PMR4-dependent. In live-cell imaging, tagged RabA4c was shown to localize at the plasma membrane prior to infection, which was broken in the pmr4 disruption mutant background, with callose deposits at the site of attempted fungal penetration. Together with our interactions studies including yeast two-hybrid, pull-down, and in planta fluorescence resonance energy transfer assays, we concluded that RabA4c directly interacts with PMR4, which can be seen as an effector of this GTPase.
The tomato—Pseudomonas syringae pv. tomato (Pst)—pathosystem is one of the best understood models for plant-pathogen interactions. Certain wild relatives of tomato express two closely related members of the same kinase family, Pto and Fen, which recognize the Pstvirulence protein AvrPtoB and activate effector-triggered immunity (ETI). AvrPtoB, however, contains an E3 ubiquitin ligase domain in its carboxyl terminus which causes degradation of Fen and undermines its ability to activate ETI. In contrast, Pto evades AvrPtoB-mediated degradation and triggers ETI in response to the effector. It has been reported recently that Pto has higher kinase activity than Fen and that this difference allows Pto to inactivate the E3 ligase through phosphorylation of threonine-450 (T450) in AvrPtoB. Here we show that, in contrast to Fen which can only interact with a single domain proximal to the E3 ligase of AvrPtoB, Pto binds two distinct domains of the effector, the same site as Fen and another N-terminal domain. In the absence of E3 ligase activity Pto binds to either domain of AvrPtoB to activate ETI. However, the presence of an active E3 ligase domain causes ubiquitination of Pto that interacts with the domain proximal to the E3 ligase, identical to ubiquitination of Fen. Only when Pto binds its unique distal domain can it resist AvrPtoB-mediated degradation and activate ETI. We show that phosphorylation of T450 is not required for Pto-mediated resistance in vivo and that a kinase-inactive version of Pto is still capable of activating ETI in response to AvrPtoB. Our results demonstrate that the ability of Pto to interact with a second site distal to the E3 ligase domain in AvrPtoB, and not a higher kinase activity or T450 phosphorylation, allows Pto to evade ubiquitination and to confer immunity to Pst.
Advanced genome-editing techniques have been used to create a strain of wheat resistant to a destructive fungal pathogen — called powdery mildew — that is a major bane to the world's top food source, according to scientists at one of China's leading centers for agricultural research.
To stop the mildew, researchers at the Chinese Academy of Sciences deleted genes that encode proteins that repress defenses against the mildew. The work promises to someday make wheat more resistant to the disease, which is typically controlled through the heavy use of fungicides. It also represents an important achievement in using genome editing tools to engineer food crops without inserting foreign genes — a flashpoint for opposition to genetically modified crops.
The gene-deletion trick is particularly tough to do in wheat because the plant has three genomes — with largely similar copies of the same genes — meaning all three must be deleted or the trait will not be changed. Using gene-editing tools known as TALENs and CRISPR, the researchers were able to do that without changing anything else or adding genes from other organisms.
"We now caught all three copies, and only by knocking out all three copies can we get this [mildew]-resistant phenotype," says Caixia Gao, who heads a gene-editing research group at the State Key Laboratory of Plant Cell and Chromosome Engineering at the Institute of Microbiology in Beijing.
Nep-1 Like Proteins (NLPs) are best known for their cytotoxic activity in dicot plants. NLPs are taxonomically widespread among microbes with very different lifestyles. To learn more about this enigmatic protein family we analyzed more than 500 available NLP protein sequences from fungi, oomycetes, and bacteria. Phylogenetic clustering showed that, besides the previously documented two types, an additional more divergent third NLP type could be distinguished. By closely examining the three NLP types, we identified a non-cytotoxic subgroup of type 1 NLPs (designated type 1a), which have substitutions in amino acids making up a cation-binding pocket that is required for cytotoxicity. Type 2 NLPs were found to contain a putative calcium-binding motif, which was shown to be required for cytotoxicity. Members of both type 1 and type 2 NLPs were found to possess additional cysteine residues that, based on their predicted proximity, make up potential disulfide bridges that could provide additional stability to these secreted proteins. Type 1 and type 2 NLPs, although both cytotoxic to plant cells, differ in their ability to induce necrosis when artificially targeted to different cellular compartments in planta, suggesting they have different mechanisms of cytotoxicity.
The basidiomycete smut fungus Ustilago hordei was previously shown to comprise isolates that are avirulent on various barley host cultivars. Through genetic crosses we had revealed that a dominant avirulence locus UhAvr1 which triggers immunity in barley cultivar Hannchen harboring resistance gene Ruh1, resided within an 80-kb region. DNA sequence analysis of this genetically delimited region uncovered the presence of 7 candidate secreted effector proteins. Sequence comparison of their coding sequences among virulent and avirulent parental and field isolates could not distinguish UhAvr1 candidates. Systematic deletion and complementation analyses revealed that UhAvr1 is UHOR_10022 which codes for a small effector protein of 171 amino acids with a predicted 19 amino acid signal peptide. Virulence in the parental isolate is caused by the insertion of a fragment of 5.5 kb with similarity to a common U. hordei transposable element (TE), interrupting the promoter of UhAvr1 and thereby changing expression and hence recognition of UhAVR1p. This rearrangement is likely caused by activities of TEs and variation is seen among isolates. Using GFP-chimeric constructs we show that UhAvr1 is induced only in mated dikaryotic hyphae upon sensing and infecting barley coleoptile cells. When infecting Hannchen, UhAVR1p causes local callose deposition and the production of reactive oxygen species and necrosis indicative of the immune response. UhAvr1 does not contribute significantly to overall virulence. UhAvr1 is located in a cluster of ten effectors with several paralogs and over 50% of TEs. This cluster is syntenous with clusters in closely-related U. maydis and Sporisorium reilianum. In these corn-infecting species, these clusters harbor however more and further diversified homologous effector families but very few TEs. This increased variability may have resulted from past selection pressure by resistance genes since U. maydis is not known to trigger immunity in its corn host.
To confer resistance against pathogens and pests in plants, typically dominant resistance genes are deployed. However, because resistance is based on recognition of a single pathogen-derived molecular pattern these narrowspectrum genes are usually readily overcome. Disease arises from a compatible interaction between plant and pathogen. Hence, altering a plant gene that critically facilitates compatibility could provide a more broad-spectrum and durable type of resistance. Here, such susceptibility (S) genes are reviewed with a focus on the mechanisms underlying loss of compatibility. We distinguish three groups of S genes acting during different stages of infection: early pathogen establishment, modulation of host defenses, and pathogen sustenance. The many examples reviewed here show that S genes have the potential to be used in resistance breeding. However, because S genes have a function other than being a compatibility factor for the pathogen, the side effects caused by their mutation demands a one-by-one assessment of their usefulness for application.
The type VI secretion system (T6SS) is a widespread molecular weapon deployed by many Proteobacteria to target effectors/toxins into both eukaryotic and prokaryotic cells. We report that Agrobacterium tumefaciens, a soil bacterium that triggers tumorigenesis in plants, produces a family of type VI DNase effectors (Tde) that are distinct from previously known polymorphic toxins and nucleases. Tde exhibits an antibacterial DNase activity that relies on a conserved HxxD motif and can be counteracted by a cognate immunity protein, Tdi. In vitro, A. tumefaciens T6SS could kill Escherichia coli but triggered a lethal counterattack by Pseudomonas aeruginosa upon injection of the Tde toxins. However, in an in planta coinfection assay, A. tumefaciens used Tde effectors to attack both siblings cells and P. aeruginosa to ultimately gain a competitive advantage. Such acquired T6SS-dependent fitness in vivo and conservation of Tde-Tdi couples in bacteria highlights a widespread antibacterial weapon beneficial for niche colonization
Movement of RNAs between cells of a single plant is well documented, but cross-species RNA transfer is largely unexplored. Cuscuta pentagona (dodder) is a parasitic plant that forms symplastic connections with its hosts and takes up host messenger RNAs (mRNAs). We sequenced transcriptomes of Cuscuta growing on Arabidopsis and tomato hosts to characterize mRNA transfer between species and found that mRNAs move in high numbers and in a bidirectional manner. The mobile transcripts represented thousands of different genes, and nearly half the expressed transcriptome of Arabidopsis was identified in Cuscuta. These findings demonstrate that parasitic plants can exchange large proportions of their transcriptomes with hosts, providing potential mechanisms for RNA-based interactions between species and horizontal gene transfer.
Two weeks ago, at a conference in South Africa, scientists met to discuss how to contain a deadly banana disease outbreak in nearby Mozambique, Africa. At fault was a fungus that continues its march around the planet. In recent years, it has spread across Asia and Australia, devastating plants there that bear the signature yellow supermarket fruit.
The international delegation of researchers shared their own approaches to the malady, hoping to arrive at some strategy to insulate Mozambique and the rest of Africa: a continent where bananas are essential to the lives of millions. They left the Cape Town-based meeting with an air of optimism.
Only days after the meeting, however, a devastating new survey of the stricken Mozambique farm was released. Scientists at the conference assumed that the fungus was limited to a single plot. The new report suggested the entire plantation was infested, expanding 125 diseased acres to more than 3,500. All told, 7 million banana plants were doomed to wilt and rot.
“The future looks bleak,” says Altus Viljoen, the South African plant pathologist who organized the conference. "There’s no way they’ll be able to stop any further spread if they continue to farm.” Worse, he says, the disease's rapid spread endangers banana crops beyond Mozambique’s borders.
The story of the African farm is the story of a threat to the world’s largest fruit crop. Commercially, bananas generate $8 billion annually and, according to the United Nations Conference on Trade and Development, more than 400 million people rely on the fruit as their primary source of calories. Though more bananas are grown in Asia, Africans depend heavily on the crop; in countries like Rwanda and Uganda, for example, average banana consumption is about 500 pounds per person annually, or 20 times that of the typical American. If the bananas vanish, people starve.
The 1000 Fungal Genome (1KFG) project is a large-scale community sequencing project supported by the Joint Genome Institute (JGI). The goal of 1KFG is to facilitate the sequencing of fungal genomes across the Kingdom Fungi with the objective to significantly advance genome-enabled mycology. The sampling guideline is to sequence two species of fungi for every family-level clade of Fungi so that genomic data is representative of phylogenetic diversity of Fungi. In support of this endeavor, 1KFG is pleased to announce the Graduate Student/Postdoc Challenge. From July 2014-June 30 2015 we will accept nominations to sequence up to 100 species of fungi in support of graduate student and postdoctoral research projects. Students and postdocs are encouraged to nominate species and submit DNA and RNA samples for genomic sequencing.
Follow the link to find out how to nominate species.
Mutualistic symbioses between eukaryotes and beneficial microorganisms of their microbiome play an essential role in nutrition, protection against disease, and development of the host. However, the impact of beneficial symbionts on the evolution of host genomes remains poorly characterized. Here we used the independent loss of the most widespread plant–microbe symbiosis, arbuscular mycorrhization (AM), as a model to address this question. Using a large phenotypic approach and phylogenetic analyses, we present evidence that loss of AM symbiosis correlates with the loss of many symbiotic genes in the Arabidopsis lineage (Brassicales). Then, by analyzing the genome and/or transcriptomes of nine other phylogenetically divergent non-host plants, we show that this correlation occurred in a convergent manner in four additional plant lineages, demonstrating the existence of an evolutionary pattern specific to symbiotic genes. Finally, we use a global comparative phylogenomic approach to track this evolutionary pattern among land plants. Based on this approach, we identify a set of 174 highly conserved genes and demonstrate enrichment in symbiosis-related genes. Our findings are consistent with the hypothesis that beneficial symbionts maintain purifying selection on host gene networks during the evolution of entire lineages.
Filamentous pathogens pose a substantial threat to global food security. One central question in plant pathology is how pathogens cause infection and manage to evade or suppress plant immunity to promote disease. With many technological advances over the past decade, including DNA sequencing technology, an array of new tools has become embedded within the toolbox of next-generation plant pathologists. By employing a multidisciplinary approach plant pathologists can fully leverage these technical advances to answer key questions in plant pathology, aimed at achieving global food security. This review discusses the impact of: cell biology and genetics on progressing our understanding of infection structure formation on the leaf surface; biochemical and molecular analysis to study how pathogens subdue plant immunity and manipulate plant processes through effectors; genomics and DNA sequencing technologies on all areas of plant pathology; and new forms of collaboration on accelerating exploitation of big data. As we embark on the next phase in plant pathology, the integration of systems biology promises to provide a holistic perspective of plant–pathogen interactions from big data and only once we fully appreciate these complexities can we design truly sustainable solutions to preserve our resources.
Plant resistance (R) genes are a crucial component in plant defence against pathogens1. Although R genes often fail to provide durable resistance in an agricultural context, they frequently persist as long-lived balanced polymorphisms in nature2, 3, 4. Standard theory explains the maintenance of such polymorphisms through a balance of the costs and benefits of resistance and virulence in a tightly coevolving host–pathogen pair5, 6. However, many plant–pathogen interactions lack such specificity7. Whether, and how, balanced polymorphisms are maintained in diffusely interacting species8 is unknown. Here we identify a naturally interacting R gene and effector pair in Arabidopsis thaliana and its facultative plant pathogen, Pseudomonas syringae. The protein encoded by the R gene RPS5 recognizes an AvrPphB homologue (AvrPphB2) and exhibits a balanced polymorphism that has been maintained for over 2 million years (ref. 3). Consistent with the presence of an ancient balanced polymorphism, the R gene confers a benefit when plants are infected with P. syringae carrying avrPphB2 but also incurs a large cost in the absence of infection. RPS5alleles are maintained at intermediate frequencies in populations globally, suggesting ubiquitous selection for resistance. However, the presence of P. syringae carrying avrPphB is probably insufficient to explain the RPS5 polymorphism. First, avrPphB homologues occur at very low frequencies in P. syringae populations on A. thaliana. Second, AvrPphB only rarely confers a virulence benefit to P. syringae on A. thaliana. Instead, we find evidence that selection for RPS5 involves multiple non-homologous effectors and multiple pathogen species. These results and an associated model suggest that the R gene polymorphism in A. thaliana may not be maintained through a tightly coupled interaction involving a single coevolved R gene and effector pair. More likely, the stable polymorphism is maintained through complex and diffuse community-wide interactions.
Sequence-specific nucleases have been applied toengineer targeted modifications in polyploid genomes, but simultaneous modification of multiple homoeoalleles has not been reported. Here we use transcription activator–like effector nuclease (TALEN) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 technologies in hexaploid bread wheat to introduce targeted mutations in the three homoeoalleles that encode MILDEW- RESISTANCE LOCUS (MLO) proteins. Genetic redundancy has prevented evaluation of whether mutation of all three MLO alleles in bread wheat might confer resistance to powdery mildew, a trait not found in natural populations. We show that TALEN-induced mutation of all three TaMLO homoeologs in the same plant confers heritable broad-spectrum resistanceto powdery mildew. We further use CRISPR-Cas9 technologyto generate transgenic wheat plants that carry mutations inthe TaMLO-A1 allele. We also demonstrate the feasibility of engineering targeted DNA insertion in bread wheat through nonhomologous end joining of the double-strand breaks caused by TALENs. Our findings provide a methodological framework to improve polyploid crops.
Scientists say an outbreak of beet western yellows virus is one of the worst cases ever seen in Australia.
Early estimates suggest up to 10,000 hectares of canola have been affected, in South Australia's lower north, mid north and lower mallee regions. The virus is transported by green peach aphids, which have thrived in the state's recent warm and humid weather. Ag consultant Mick Faulkner says agronomists felt like they'd been "blind-sided" after not being able to work out what had been affecting crops. "It took everyone a fair bit of time to realise that we weren't killing the aphids," Mr Faulkner said. "Green paddocks are now brown. "Those that have been affected, I have grave fears that they won't yield anything at all."
Virus halted for now - The South Australian Research and Development Institute (SARDI) says it's now testing samples to confirm how the virus is spreading and where else it might turn up. Pulse pathologist Jenny Davidson says with cooler weather, the virus-transmitting aphids aren't moving and at the moment the best thing growers can do is "nothing", "We expect that the spread of this virus would've stopped for now, so there's no point people going out and spraying aphids now," she says. "It's also important growers ascertain it actually is the virus causing problems in their canola crops, there may be other things going on as well. "The potential risk is what these aphids will do in spring time. "We're not sure whether or not pulse crops are at risk but we'll have that information back well and truly before the spring time flights." Ms Davidson says the virus isn't uncommon, but what is unusual is the extent of damage and infection being seen. She says it's taken everyone by surprise. "I've never seen this level of damage from any virus in crops," Ms Davidson says. "It's the magnitude of what we're dealing with that is totally un-expected."
Plant resistance proteins of the class of nucleotide-binding and leucine-rich repeat domain proteins (NB-LRRs) are immune sensors which recognize pathogen-derived molecules termed avirulence (AVR) proteins. We show that RGA4 and RGA5, two NB-LRRs from rice, interact functionally and physically to mediate resistance to the fungal pathogen Magnaporthe oryzae and accomplish different functions in AVR recognition. RGA4 triggers an AVR-independent cell death that is repressed in the presence of RGA5 in both rice protoplasts and Nicotiana benthamiana. Upon recognition of the pathogen effector AVR-Pia by direct binding to RGA5, repression is relieved and cell death occurs. RGA4 and RGA5 form homo- and hetero-complexes and interact through their coiled-coil domains. Localization studies in rice protoplast suggest that RGA4 and RGA5 localize to the cytosol. Upon recognition of AVR-Pia, neither RGA4 nor RGA5 is re-localized to the nucleus. These results establish a model for the interaction of hetero-pairs of NB-LRRs in plants: RGA4 mediates cell death activation, while RGA5 acts as a repressor of RGA4 and as an AVR receptor.
Terrestrial plants rely on stomata, small pores in the leaf surface, for photosynthetic gas exchange and transpiration of water. The stomata, formed by a pair of guard cells, dynamically increase and decrease their volume to control the pore size in response to environmental cues. Stresses can trigger similar or opposing movements: for example, drought induces closure of stomata, whereas many pathogens exploit stomata and cause them to open to facilitate entry into plant tissues. The latter is an active process as stomatal closure is part of the plant's immune response. Stomatal research has contributed much to clarify the signalling pathways of abiotic stress, but guard cell signalling in response to microbes is a relatively new area of research. In this article, we discuss present knowledge of stomatal regulation in response to microbes and highlight common points of convergence, and differences, compared to stomatal regulation by abiotic stresses. We also expand on the mechanisms by which pathogens manipulate these processes to promote disease, for example by delivering effectors to inhibit closure or trigger opening of stomata. The study of pathogen effectors in stomatal manipulation will aid our understanding of guard cell signalling.