MicrobiologyBytes

Strategies in modern agriculture aim to enhance harvest yields per acreage and to reduce pre-harvest and post-harvest losses caused by detrimental abiotic and biotic causes. The potential of that reflects an estimate 20–40% reduction of agricultural production worldwide that is taken by pests and diseases. Modern pest management strategies in crop plants include classical and molecular marker-based resistance breeding, genetic engineering of plant immunity and the use of chemicals as pesticides or strengtheners of plant health. While breeding strategies are time-consuming and harbor the problem of ‘linkage drag’ (transfer of undesirable traits that need to be removed after introgression of the desired trait by back-crossing), genetic engineering holds the potential of being reasonably fast and predictable in its consequences because of the targeted introduction of individual, heterologous traits into elite crop lines.

Sequencing of entire plant genomes, systematic plant transcriptome profiling and comprehensive genetic dissection of immune pathways in model plants (Arabidopsis thaliana, rice) has significantly enhanced our understanding of the mechanisms underlying microbial infection and plant immunity. The plant immune system consists of two evolutionarily linked branches. Recognition of invariant microbial surface patterns (pathogen or microbe-associated patterns; PAMP/MAMP) through plant pattern recognition receptors is referred to as PAMP-triggered immunity (PTI) and is the basis for broad-spectrum resistance of plants against host non-adapted microbial pathogens (i.e. all genetic variants of a given microbial species are unable to grow on a given plant species).

Novel insight into plant immunity and disease may now be turned into new tools to engineer durable, broad-spectrum plant disease resistance. This review highlights recent scientific discoveries in plant immunity and discusses their potential for enhancing plant immunity in crop plants with particular emphasis on immunity to bacterial and fungal infection. Saving the world’s food supply constitutes one of the major challenges of the future. As a complement to classical and molecular breeding technologies, novel strategies for biotechnological improvement of plant immunity aim at enhancing host recognition capacities for potential pathogens, at boosting the executive arsenal of plant immunity, and at interfering with virulence strategies employed by microbial pathogens. In addition, chemical and biological priming provides means for triggering plant defenses in a non-transgenic manner. Major advances in our understanding of the molecular basis of plant immunity and of microbial infection strategies have opened new ways for engineering durable disease resistance in crop plants that are highlighted in this review.

Biotechnological concepts for improving plant innate immunity. Curr Opin Biotechnol. Feb 22 2010

Related:

• Toxins for microbial attack and plant defence
• Resistance to Plant Viruses
• Bacteriophages for plant disease control

Pepper Mild Mottle Virus, a Plant Virus Associated with Specific Immune Responses, Fever, Abdominal Pains, and Pruritus in Humans. 2010 PLoS ONE 5(4): e10041. doi:10.1371/journal.pone.0010041
Recently, metagenomic studies have identified viable Pepper mild mottle virus (PMMoV), a plant virus, in the stool of healthy subjects. However, its source and role as pathogen have not been determined.
Methods and Findings: 21 commercialized food products containing peppers, 357 stool samples from 304 adults and 208 stool samples from 137 children were tested for PMMoV using real-time PCR, sequencing, and electron microscopy. Anti-PMMoV IgM antibody testing was concurrently performed. A case-control study tested the association of biological and clinical symptoms with the presence of PMMoV in the stool. Twelve (57%) food products were positive for PMMoV RNA sequencing. Stool samples from twenty-two (7.2%) adults and one child (0.7%) were positive for PMMoV by real-time PCR. Positive cases were significantly more likely to have been sampled in Dermatology Units (p<10−6), to be seropositive for anti-PMMoV IgM antibodies (p = 0.026) and to be patients who exhibited fever, abdominal pains, and pruritus (p = 0.045, 0.038 and 0.046, respectively).
Conclusions:
Our study identified a local source of PMMoV and linked the presence of PMMoV RNA in stool with a specific immune response and clinical symptoms. Although clinical symptoms may be imputable to another cofactor, including spicy food, our data suggest the possibility of a direct or indirect pathogenic role of plant viruses in humans.

Not so fast!

New Scientist reports suggests because the researchers looked at many possible symptoms, they would be expected to find a few that randomly appear more common in virus-positive people. In order to enter a cell and replicate, a virus must bind to a receptor on its surface, and a plant virus would be highly unlikely to recognise a receptor on a human cell.

What do you think?

The RNA silencing based antiviral plant response is one of the best studied antiviral strategies in plants. The key element of RNA silencing based antiviral strategies is the virus derived small interfering RNA (vsiRNA), which guides the RNA induced silencing complex (RISC) to target viral genomes in plants and invertebrates. siRNAs are processed from double-stranded RNAs (dsRNA) or structured single-stranded RNAs (ssRNAs) by RNase III-like enzymes such as DICER (in plants there are several Dicer-like genes). siRNAs guide the sequence-specific inactivation of target mRNAs by RISC. Plant RNA viruses are strong inducers as well as targets of RNA silencing and high levels of vsiRNAs accumulate during the viral infection. However, despite of the extensive studies of siRNA biogenesis the origin of plant viral siRNA is still not understood. vsiRNAs are thought to be processed from ds viral RNA replication intermediates, local self-complementary ds regions of the viral genome or through the action of RNA-dependent RNA polymerases on viral RNA templates. In plants two distinct classes of vsiRNAs have been identified: the primary siRNAs, which result from DCL mediated cleavage of an initial trigger RNA, and secondary siRNAs, whose biogenesis requires an RDR enzyme.

Researchers profiled Cymbidium ringspot virus (CymRSV) derived short RNAs using three different methods. Profiling of viral short interfering RNAs revealed a different sequence bias for the 454 and Solexa high-throughput sequencing platforms. They found that viral short RNAs are primarily produced from the positive strand of the virus and produced with very different frequency along the viral genome. The hybridisation approach showed that the profile of viral short RNAs is determined by the virus itself because the profiles were the same in different species and it also showed that the process was RDR6 independent. These results suggest that CymRSV short RNAs are produced from the structured positive strand rather than from perfect double stranded RNA or by RNA dependent RNA polymerase. Regions from the viral genome that are not complementary to highly abundant viral short RNAs were targeted in the plant just as efficiently as regions recognised by abundant short RNAs.

Structural and Functional Analysis of Viral siRNAs. 2010 PLoS Pathog 6(4): e1000838. doi:10.1371/journal.ppat.1000838
A large amount of short interfering RNA (vsiRNA) is generated from plant viruses during infection, but the function, structure and biogenesis of these is not understood. We profiled vsiRNAs using two different high-throughput sequencing platforms and also developed a hybridisation based array approach. The profiles obtained through the Solexa platform and by hybridisation were very similar to each other but different from the 454 profile. Both deep sequencing techniques revealed a strong bias in vsiRNAs for the positive strand of the virus and identified regions on the viral genome that produced vsiRNA in much higher abundance than other regions. The hybridisation approach also showed that the position of highly abundant vsiRNAs was the same in different plant species and in the absence of RDR6. We used the Terminator 5′-Phosphate-Dependent Exonuclease to study the 5′ end of vsiRNAs and showed that a perfect control duplex was not digested by the enzyme without denaturation and that the efficiency of the Terminator was strongly affected by the concentration of the substrate. We found that most vsiRNAs have 5′ monophosphates, which was also confirmed by profiling short RNA libraries following either direct ligation of adapters to the 5′ end of short RNAs or after replacing any potential 5′ ends with monophosphates. The Terminator experiments also showed that vsiRNAs were not perfect duplexes. Using a sensor construct we also found that regions from the viral genome that were complementary to non-abundant vsiRNAs were targeted in planta just as efficiently as regions recognised by abundant vsiRNAs. Different high-throughput sequencing techniques have different reproducible sequence bias and generate different profiles of short RNAs. The Terminator exonuclease does not process double stranded RNA, and because short RNAs can quickly re-anneal at high concentration, this assay can be misleading if the substrate is not denatured and not analysed in a dilution series. The sequence profiles and Terminator digests suggest that CymRSV siRNAs are produced from the structured positive strand rather than from perfect double stranded RNA or by RNA dependent RNA polymerase.

Related:

• Resistance to Plant Viruses
• A cream that could stop genital herpes?

A mycorrhiza is a symbiotic association between a fungus and the roots of a plant. In a mycorrhizal association, the fungus may colonize the roots of a host plant, either intracellularly (arbuscular mycorrhizal fungi) or extracellularly (ectomycorrhizal fungi). These communities are important in plant growth and soil flora. This review focuses on interactions among plants, mycorrhizal fungi, and bacteria, testing the hypothesis whether mycorrhizas can be defined as tripartite associations. After summarizing the main biological features of mycorrhizas, it illustrates the different types of interaction occurring between mycorrhizal fungi and bacteria, from loosely associated microbes to endobacteria. It also discusses, in the context of nutritional strategies, the mechanisms that operate among members of the consortium and that often promote plant growth. Release of active molecules, including volatiles, and physical contact among the partners seem important for the establishment of the bacteria/mycorrhizal fungus/plant network. The potential involvement of quorum sensing and Type III secretion systems is discussed, even if the exact nature of the complex interspecies/interphylum interactions remains unclear.

Plants, mycorrhizal fungi, and bacteria: a network of interactions. Ann Rev Microbiol. 2009 63: 363-83

Related:

• Evolution of root nodule symbiosis with nitrogen-fixing bacteria

Recent research has revealed that some plant viruses, like many animal viruses, have measurably evolving populations. Most of these viruses have single-stranded positive-sense RNA genomes, but a few have single-stranded DNA genomes. The studies show that extant populations of these virus species are only decades to centuries old, and the genera in which they are placed have diverged since agriculture was invented, and spread around the world during the Holocene. We suggest that this is not mere coincidence but evidence that the conditions generated by agriculture during this era have favoured particular viruses. There is also evidence, albeit less certain, that some plant viruses, including a few shown to have measurably evolving populations, have much more ancient origins. We discuss the possible reasons for this clear discordance between short-term and long-term evolutionary rate estimates, and how it might result from a large timescale dependence of the evolutionary rates. We also discuss briefly why it is useful to know the rates of evolution of plant viruses.

Time – the emerging dimension of plant virus studies. J Gen Virol. Nov 4 2009

Related:

• Plant viruses: master explorers of evolutionary space. Curr Opin Genet Dev. 1993 3(6): 873-877
• Experimental evolution of plant RNA viruses. Heredity. 2008 100(5): 478-483
• Resistance to Plant Viruses

Millions of years of coevolution of plants and microbial pathogens have shaped both the abilities of microbial pathogens to overcome plant disease resistance and the abilities of plants to cope with microbial invasion. Phytopathogens from different taxonomic origins secrete structurally unrelated effectors into plants to establish infection and to suppress host defences. In addition, phytopathogenic micro-organisms produce a wide range of cytolytic toxins that function as virulence determinants.

Microbial pattern recognition is a prerequisite for the initiation of antimicrobial defenses in all multicellular organisms, including plants. The bipartite plant immune system is based upon recognition of pathogen-associated molecular patterns by pattern-recognition receptors as well as upon the activities of resistance proteins that have evolved to recognize the presence or activities of microbial effectors. In addition to the recognition of microbial patterns and effectors, plants also possess capacities to sense host-derived damage patterns that originate, for example, from the degradation of the plant cell wall by microbial hydrolytic enzymes.

Paradoxically, some phytopathogenic microbe-derived cytolytic toxins have also been reported to elicit plant defences. However, for virtually all microbial toxins with plant defence-stimulating potential, it is unknown whether activation of plant defences results from toxin-induced cellular distress or, independently of toxin action, from recognition of toxins as microbial patterns by plant pattern-recognition receptors.

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NLPs are a superfamily of proteins that are produced by various phytopathogenic micro-organisms, both prokaryotes and eukaryotes. Necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) trigger leaf necrosis that is genetically distinct from immunity-associated programmed cell death and stimulate immunity associated defences in all dicotyledonous plants tested, but not in monocotyledons such as grasses. Hence, NLPs were proposed to have dual functions in plant pathogen interactions, acting both as triggers of immune responses and as toxin-like virulence factors. The broad taxonomic distribution of NLPs, in particular their occurrence in both prokaryotic and eukaryotic species, is unusual for known microbial phytotoxins, the production of which is restricted to a narrow range of microbial species.

Recent work has determined the crystal structure of an NLP from a phytopathogenic fungus (A common toxin fold mediates microbial attack and plant defense. PNAS USA June 11 2009, doi: 10.1073/pnas.0902362106). Computational modeling of the three-dimensional structure of NLPs from another fungus and from a phytopathogenic bacterium reveals a high degree of conservation. Expression of the fungus NLPs in an NLP-deficient phytopathogenic bacteria restored bacterial virulence.

Mutation analysis revealed that identical structural properties were required to cause plasma membrane permeabilization and cytolysis in plant cells, as well as to restore bacterial virulence. The conclusion is that NLPs are conserved virulence factors whose wide taxonomic distribution is exceptional for microbial phytotoxins, and that contribute to host infection by plasma membrane destruction and cytolysis. Phytotoxin-induced cellular damage-associated activation of plant defenses is reminiscent of microbial toxin-induced inflammatory activation in vertebrates and may constitute another conserved element in animal and plant innate immunity.

Related:

• Toll-Like Receptors
• Bacterial toxins

Some estimates put total the worldwide economic damage due to plant viruses as high as US$60 billion per year, in addition to the human costs in terms of hunger and poverty. So understanding how viruses damage plants and how this can be avoided is of the utmost importance. There are hundreds of plant-pathogenic viruses, which cause a range of diseases. However, plants have evolved elaborate and effective defence mechanisms to prevent or limit virus damage. Plants contain resistance (R) genes, which allow resistance to a range of pathogens including viruses. Each R gene gives resistance to a particular pathogen. A number of R genes have been studied in detail (Mechanisms of plant resistance to viruses. 2005 Nature Reviews Microbiology 3: 789-798). Several molecules and signalling pathways are induced on pathogen recognition, and they cooperate to produce a defensive response. Some of the best characterized of these molecules include salicylic acid, nitric oxide and reactive oxygen species, in addition to some plant hormones. RNA silencing is a highly conserved pathway in animals and plants that functions in development and in the maintenance of genome integrity. Plants have adapted this system for antiviral defences, and of course, plant viruses have in turn developed mechanisms to suppress RNA silencing. Double-stranded RNA (dsRNA) is the trigger for RNA silencing. Most plant viruses (59 of the 80-odd plant virus genera) are RNA viruses and plants have several homologues of the DICER endonuclease. These enzymes generate siRNA (short interfering RNA) as an antiviral response. So these two pathways – RNA silencing and R-gene-mediated resistance – interact to produce an effective defence response in plants.

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Having penetrated the plant cell wall, the first defence mechanism which viruses encounter are extracellular surface receptors on the host cell plasma membrane that recognize pathogen-associated molecular patterns (PAMPs). This interaction initiates PAMP-triggered immunity (PTI), which sometimes halts infection before the virus gains a hold in the plant. However, plant viruses have evolved the means to suppress PTI by interfering with recognition at the plasma membrane. Effector-triggered immunity involves the direct or indirect recognition of the virus proteins used to subvert PTI by plant R proteins (Host-Microbe Interactions: Shaping the Evolution of the Plant Immune Response. 2006 Cell 124: 803-814). Virus virulence determinants suppress the host RNA silencing response.

Genetic engineering offers a means of incorporating new virus resistance traits into existing plant varieties. The initial attempts to create transgenes conferring virus resistance were based on the pathogen-derived resistance determinants, for example, the expression of virus coat protein genes in transgenic plants was shown to induce protective effects. Since then, a large variety of virus genes encoding structural and non-structural proteins has also been shown to confer resistance to disease (Strategies for antiviral resistance in transgenic plants. 2008 Molecular Plant Pathology 9: 73-83). Subsequently, non-coding virus RNAs have been shown to be a potential trigger for virus resistance in transgenic plants, which led to the discovery of RNA silencing.

Plants have evolved a robust innate immune system that exhibits striking similarities as well as significant differences with innate immunity in animals. For example, plants are capable of perceiving PAMPs through pattern recognition receptors that bear structural similarities to animal Toll-like receptors. In addition, plants have evolved a second surveillance system based on cytoplasmic “NB-LRR” proteins (nucleotide-binding, leucine-rich repeat) that are structurally similar to animal nucleotide-binding and oligomerization domain (NOD)-like receptors. Plant NB-LRR proteins do not detect PAMPs, but recognize proteins that viruses produce in plant cells. (Molecular diversity at the plant-pathogen interface. 2007 Dev Comp Immunol)

The number of different strategies that have been developed for creating virus and viroid resistance is one of the major success stories in biotechnology. However, few of these strategies have ever been taken past the proof of principle stage in the laboratory, or small-scale field trials. The only engineered virus-resistant plants that have been grown on a large commercial scale were transformed with complete virus transgenes, and it appears that the resistance induced is of the RNA silencing type in all these cases. That means that there is still enormous potential for overcoming the detrimental effects of plant viruses through genetic manipulation of valuable plant varieties.