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Download Lectures 15-16 Molecular mechanisms of plant
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Lectures 15-16 Molecular mechanisms of plant-pathogen interactions During the contact between plant and pathogen, a particular chain of events is produced in the plant organism. Two ways of possible interaction between plant and pathogen • The plant is provided by a receptor that interacts with bacterial protein. As a result, quick protective reaction is being developed. In such a situation, the bacteria is called avirulent for a given plant genotype (Piffanelli P. et al., 1999, Martin G.B., 1999). •• The proteins of the pathogenic organism are virulent for the given plant genotype. The plant is affected by the pathogen, whereas protective mechanisms are being activated more slowly (Maleck K and Lawton K., 1998). In both cases, with the start of pathogenesis gene transcription, the cell walls strengthen. Then in the place of pathogen penetration, the active forms of oxygen are formed, causing the death of infected cells. Molecular mechanisms of plant resistance to disease Plant disease caused by viruses, bacteria and fungi are controlled naturally by plant host resistance (R) genes and avirulence (avr) genes of the pathogen. This has been termed gene-for-gene resistance. A simple model explains these gene-for-gene interactions: Avirulence gene products generate signals (ligands) and resistance genes encode cognate receptors. Disease resistance is usually mediated by dominant genes, but some recessive resistance genes also exist. In early 1900s it was recognized that resistance to plant pathogens was often inherited as a single dominant or semidominant trait. 1940s – Harold Flor studied flax and flax rust pathogen. Gene-for-gene interaction. Model: Plant resistance will only occur when a plant possesses a dominant resistance gene (R ) and a pathogen expresses complementary dominant avirulence gene (Avr). Holds true for most plant-pathogen interactions.Flor’s model: For resistance(incompatibility) to occur, complementary pairs of dominant genes, one in the host one in the pathogen, are required. An alteration or loss of the plant resistance gene (R changing to r) or of the pathogen avirulence gene (Avr changing to avr) leads to disease (compatible).Different model – for pathogens that deploy host-selective toxins for successful pathogenesis. Pathogen virulence must be dominant because a functional toxin or enzyme (or both) must be produced to cause disease. Plant resistance is predominantly inherited as a dominant trait and is achieved through enzymatic detoxification or through loss or alteration of the toxin in the pathogen. Interactions involved in toxin-dependent compatibility. The wild-type pathogen gene Tox is required for synthesis of a toxin that is crucial for pathogenesis; tox is the corresponding recessive, nonfunctional allele. The dominant allele of the host R gene is required for detoxification-based resistance. Resistance can only occur when the host expresses a toxin-insensitive for of the toxin target. Disease results only when the plant can not detoxify the toxin produced by the pathogen.The term disease tolerance describes a neutral outcome observed in plant-pathogen associations. The interaction is genetically compatible, but the plant somehow restricts the biochemical processes required for symptom development. As a result tissue damage is kept down even if the plants are heavily infected. Disease-tolerant plants act as important reservoirs of pathogen inocula, which may go and infect susceptible species.Properties of some avirulence genesAvr genes are recognized as the genetic determinants of incompatibility toward particular plant genotypes. Functions of Avr genes of phytopathogenic bacteria or fungi are not completely understood. Plant viruses – conclusive exceptions. In different incompatible interactions the viral CP, replicase and MP are recognized as avirulence factors. Changes in amino acids that do not substantially compromise the primary function of protein in pathogenesis can still alter their avr specificity. A mutation from avirulence (Avr) to virulence (avr) is often associated with the loss of fitness for growth on plants that lack the R gene. Thus, Avr gene products have important roles in microbial pathogenicity at different stages: growth and reproduction in plants, symptom development, transfer to other plants.1984 – the first avirulence gene was isolated from Pseudomonas bacteria infecting soybean. Later 30 avr genes were isolated from Pseudomonas and Xanthomonas species. Bacterial avr genes code for soluble, hydrophylic proteins. Avr proteins share little homology with other known proteins, but there is substantial homology between some Avr proteins. Two types of distinct Avr-generated signals are recognized. Exported syringolides (Cglucosides) are produced by enzymes encoded by the avrD locus of P. syringae pv. glycinea, and these substances trigger a resistance response in soybean harboring corresponding R gene, Rpg4. For other bacterial species, the Avr protein itself is now thought to be a signal.The Xanthomonas avrBs3 family of avr genes is distinct from the Pseudomonas genes. avrBs3 family proteins contain a reiterated internal motif 34 aa lonf. Eg: AvrBs3 gene product has 17.5 nearly identical repeats of this motif. By deleting some of these repeats the specificity of this Avr gene can be altered, so that it is no longer recognized by the corresponding Bs3 resistance gene in pepper plants. Other internal deletions in the AvrBs3 lead to the gain-of-function phenotype, resulting in resistance response in plant carrying recessive bs3 allele.Delivery of AvrBs3 in to the plant cell cytoplasm via the type III secretion system appears to be necessary for the function of the Xanthomonas Avr gene products. Mutational studies of the two functional nuclear localization signals in the C-terminus of the AvrBs3 protein reveal that the Avr protein has to be targeted to the plant cell nucleus for Bs3-mediated resistance to operate. In fungi only few Avr genes are known. For fungal species that colonize only the intercellular airspaces of plants, a biochemical approach can isolate small secreted peptides capable of eliciting R-gene dependent plant defense in the absence of pathogens. They appear to be direct elicitors, but their role is still elusive.Resistance (R) genesThere are many resistance (R) genes in plant hosts, each conferring a unique specificity to various pathogen isolates. These R genes often are clustered as complex gene-families in plant genomes. In general, R genes function to recognize, directly or indirectly, "elicitor" molecules produced by the invading pathogen. This recognition results in a rapid signal cascade, leading to an active defense response. R genes (and their associated responses) are exploited by plant breeders to offset yield loss due to pathogen infection R genes and R gene-mediated disease resistanceIsolation strategies: Locating R gene on the chromosome by using plant populations that segregate for resistant and susceptible individuals. Identifying the correct sequence by inserting either transposon to destroy biological activity, or by using binary cosmid complementation to confer resistant phenotype on a susceptible plant. R genes were isolated from 3 monocotyledonous plants and from 5 dicotyledonous plants. Provide resistance to a range of taxonomically unrelated pathogens. 4of 6 classes of predicted R proteins which mediate dominant or semidominant resistance, have leucine-rich repeats (LRRs) – structural motifs seen in proteins that function in signal transduction pathways. LRR motifs have been shown to mediate protein-protein or receptor-ligand interactions in many different kinds of proteins. Genetic evidence suggests that the beta-strand/beta-turn of the LRR is a key region in the R protein and appears to determine its specificity.Many proteins also have a central nucleotide-binding site (NBS) that contains several conserved domains, the function of which is still unknown. Although they do not have intrinsic kinase activity, they could bind ATP or GTP and then activate the defense response. Mutations in key residues in the NBS destroy the R protein function. Some current models view NBS as adaptor region, linking the C-terminal LRR recognition domain to various N-terminal effectors. Some R protein possess a putative leucine zipper (LZ) or coiled-coil sequence between the N terminus and the NBS domains. Lzs are known for their roles in homo- and hetero-dimerization of eukaryotic transcription factors as well as facilitating interactions between proteins with other functions. Other NBS-LRR R proteins contain a large N-terminal domain called Toll/interleukin1/resistance (TIR) domain, that is similar to the cytoplasmatic signaling domain in Drosophila How R and Avr gene products activate plant defense responses is not understoodEach R gene product is thought to possess two functions: •Recognition of corresponding Avr-derived signal •And activation of down-stream signaling pathways to trigger complex defense responses. The various predicted protein structures provide the clues of the possible mechanisms.Example: Pto kinase – confers resistance to strains of Pseudomonas that express avrPto, a protein that bacterium delivers into the cell by type II secretion system. Yeast two-hybrid technology provided clues that it may interact directly with the avrPto signal.. Several plant protein that interact with the Pto kinase were identified, including proteins that have homology with transcription factors and another protein kinase, Pti1. Three of the possible transcription factors – Pti4, Pti5 and Pti6 possess highly conserved DNAbinding domain that recognizes a core hexanucleotide sequence – a sequence foundPlant-pathogen interactionThe basic principle behind these are that there is a gene product that inhibits an effect or product of the viral infection. One gene for infection, one gene to resist. A few examples are the L6 gene for the Flax Rust virus, the N gene in tobacco for TMV resistance, as well as the RPS2 gene in Arabidopsis for bacterial resistance. These gene-for-gene resistance's operate on the basis of recognition of the infecting particle by an interaction between the host resistance system, and the avirulence genes of the infecting virus. This results in the activation of anti-pathogen molecules, or hypersensitive cell death pathways, in the tissues around the point of infection. The RPS2, and N gene products have domains for nucleotide binding, and repeats of leucine-rich area. The postulated mechanism involves a signal transduction that is dependent on A\GTP, responding to pathogen avirulence genes. Pathogen – any organism which is able to cause diseaseDisease would result in appearance of specific symptoms due to the change of host metabolism required by pathogen or as a specific host response Important definitions•Targeting of R-gene mediated resistance leads to the expression of salicylic acid (SA) ••SA expression results in hypersensitive response (HR) and systemic acquired resistance (SAR) ••HR – necrotic lesions around the infection sites due to profound burst of reactive oxygen species ••SAR – an inducible plant defence state, the activation of which depends mostly on the accumulation of SA At the present moment, the best studied are the molecular mechanisms providing hypersensitive response (HR) (Halterman D.A. and Martin G.B., 1997). In this case, the plant receptor interacts with the pathogen molecule. In order such interaction could occur, the plant and bacteria of a certain genotype should meet, i.e., a bacteria carrying the avirulence gene (avr) interacts with a plant, which has the corresponding R-gene. Such process is called an incompatible combination and leads to quick progressing of events, or to hypersensitive response. Receptors activate the passes of signal transduction and launch several protective systems. In the place of pathogen penetration, the strong oxidants are being synthesized such as H2O2, O2· - , OH· . Then the oxidative burst is being developed, followed by the death of infected cells according to the mechanism similar to apoptosis known in vertebrates (Lam E. et al., 1999). Hypersensitive response is being developed in the place of pathogen penetration into the neighboring cells. Rapidly developing local process produces signal molecules spreading along vascular system of a plant. In dependence of local events, the set of signal molecules is being organized, which in turn, forms this or that generalized response. In the whole plant organism, the pathogenesis-related genes (PR-genes) are activated, the cell walls strengthen, and the plant accumulates some amount of protective substances, which are more effective in the struggle with this definite pathogenic form. In the plant cells, the salicylic acid (SA) is produced in considerable amounts and causes activation of SA-induced genes. The integrity of these events is named as systemic acquired resistance (SAR) (Mittler R. et al., 1999). The other systemic, or referring to the whole organism, response to pathogen infection is an induced systemic resistance (ISR). Its differs from SAR by activation of some differing set of pathogenesis genes and by other ways of signal transduction (without participance of salicylic acid) (Pieterse C.M.J. et al., 1996). The plant is not always supplied with receptors to the proteins of “attacking” bacteria or fungi. In this situation, the pathogen is called viral for given plant genotype; and the pair plant-pathogen is compatible. In this case, pathogen molecules are non-specific elicitors, which are non-specific substances causing pathogenesis. The ways of obtaining the signal from non-specific elicitors are still unknown. Various external stimuli (wound, non-specific elicitors) activate protein kinases and genes of signal molecules biosynthesis. In the course of signal transduction, the synthesis of JA, NO, H2O2, SA, and ethylene is produced. The processes are being activated that are well-known in animals: proteinkinase cascade, polyubiquitine-dependent protein degradation, etc. The signal transduction paths frequently intercross. For example, the gene of one of the key enzymes, PAL (phenylalanine ammonia-lyase), is activated not only in the course of hypersensitive response, but also in response to various external stimuli (Dixon R.A. and Paiva N.L. 1995, Mauch-Mani B. et al., 1996). PAL takes part in the synthesis of SA, phytoalexins, and lignin monomers. The latter, both in hypersensitive response and non-specific pathogenesis induction, activate pathogenesis-related (PR) genes What is the role of SA? •SA plays an important role in the support of HR (transgenic plants which express the enzyme degrading SA are not resistant to TMV infection) •SA was shown to move from the infected leaf into the other parts of the plant (up to 70% of labelled SA was found in the upper leaves). ROS are often produced during the early stages of a plant resistance responseVarious PR proteins From B.Buchanan, W. Gruissen, R. Jones Part of the material adapted from B.Buchanan, W. Gruissen, R. Jones, Biochemistry and molecular biology of plants, 2000, ASPP Publisher. http://taipan.nmsu.edu/EPWS310Dr. Roger Wise, USDA-ARS-Corn Insects and Crop Genetics Research Unithttp://www.public.iastate.edu/~imagefpc/Subpages/research.html Dr. Barbara Baker http://plantbio.berkeley.edu/faculty/faculty_pages/Baker.htmlhttp://wwwmgs.bionet.nsc.r u/mgs/papers/goryachkovsky/plant-trrd/pathogen1.htm