Avirulence Factors Research Articles

Megaprimer-mediated domain swapping for construction of chimeric viruses

Clones that encode viral genomes constructed from two viruses with contrasting biological properties have been widely used in studies of viral–host interactions, particularly when the objective is to determine the identity of the viral component recognized by the host in a resistant response, known as the avirulence factor. This paper presents an efficient method based on megaprimer-mediated domain swapping for the construction of clones encoding chimeric viral genomes as a versatile and widely applicable alternative to conventional restriction enzyme digestion and ligation methods. Potato virus X (PVX)-derived vectors expressing genes encoding fluorescent proteins were used to demonstrate this concept. The cyan fluorescent protein (CFP) gene was cloned into a binary PVX vector and subsequently replaced with the yellow fluorescent protein (YFP) gene using the megaprimer amplification reaction. DNA fragments up to 1480 bp could be replaced efficiently and quickly. Most viral clones showed the expected change in phenotype without altered infectivity. Sequence analysis revealed mutations were not introduced into the four domain-swapped plasmids. This approach will provide a valuable tool for determining which domains of a viral genome are essential for infectivity, avirulence, or otherwise determine biologically significant properties of plant viruses.

Interaction of the Carlavirus Cysteine-Rich Protein with the Plant Defense System

The genome of viruses of the genus Carlavirus codes for a small cysteine-rich protein (CRP) with unknown functions. To study the role of CRP of the chrysanthemum virus B (CVB), a recombinant potato virus X (PVX) genome containing the CVB CRP gene was constructed. Expression of CVB CRP in the PVX genetic surrounding drastically changed the character of symptoms produced by PVX in Nicotiana benthamiana. The recombinant virus caused local necrotic lesions of inoculated leaves and necrosis of apical leaves rather than asymptomatic infection and mild mosaic, which are caused by PVX in this host plant. In N. tabacum, the infection pattern depended on the plant host genotype: recombinant PVX spread systemically only in plants carrying the N gene. Agroinfiltration-mediated transient expression assays showed that CRP neither acts as an avirulence factor in N. benthamiana nor suppresses posttranscriptional gene silencing. CVB CRP was identified as a virus pathogenicity determinant that controls the interaction of the virus with the host plant in a way that depends on plant defense, which is mediated by resistance genes such as the N gene.

Indirect Defenses

Some plants defend against fungal infection by using the hypersensitive response, in which plant cells at the site of invasion are killed off to retard spread of the infection. The process relies upon an elicitor from the pathogen and a corresponding resistance gene in the plant. When Cladosporium fulvum infects tomato leaves, these two factors do not, however, seem to interact directly. Rooney et al. analyze the function of the plant protease Rcr3 in mediating the defense response. The pathogen avirulence factor, Avr, is secreted extracellularly, where it interacts with the tomato plant Rcr3 protease. Together, this interaction signals to the membrane-bound host resistance factor, Cf-2, to initiate plant defense responses. H. C. E. Rooney, J. W. van 't Klooster, R. A. L. van der Hoorn, M. H. A. J. Joosten, J. D. G. Jones, P. J. G. M. de Wit, Cladosporium Avr2 inhibits tomato Rcr3 protease required for Cf-2 -dependent disease resistance. Science 308 , 1783-1786 (2005). [Abstract] [Full Text]

Constitutive expression of bvgR-repressed factors is not detrimental to the Bordetella bronchiseptica–host interaction

Bordetella bronchiseptica infection requires the activation of virulence genes by the two-component BvgAS regulatory system, which also activates bvgR, a repressor of another set of genes called avirulence genes. Whether or not BvgR-repressed genes play a role in pathogenesis is poorly understood. To evaluate their possible contribution to the bacteria–host interaction we constructed a B. bronchiseptica bvgR insertional mutant ( BbBvgR mutant). As expected, this mutant simultaneously expressed virulence and avirulence markers. In vitro experiments demonstrated that, although the BbBvgR mutant expressed avirulence factors during its virulent state, the bacteria adhered to and survived within human epithelial cells as efficiently as the wild-type strain. The mutant was not impaired for colonization of the respiratory tract in vivo, as it was effectively cleared from lungs during the same time period as the wild-type strain.

Molecular Properties of the Xanthomonas AvrRxv Effector and Global Transcriptional Changes Determined by Its Expression in Resistant Tomato Plants

The Xanthomonas campestris pv. vesicatoria avirulence gene AvrRxv specifies resistance on the tomato line Hawaii 7998 by interacting with three nondominant plant resistance genes. AvrRxv molecular properties that impinge on its avirulence activity were characterized and transcriptional changes caused by AvrRxv expression in resistant tomato plants were extensively examined. AvrRxv localized predominantly to the cytoplasm and possibly in association with plasma and nuclear membranes in both resistant and susceptible tomato plants. The AvrRxv cysteine protease catalytic core was found to be essential for host recognition, because introduction of mutations in this domain affected the ability of AvrRxv to elicit a hypersensitive response and the inhibition of bacterial growth in resistant plants. In addition, expression profiles were analyzed for approximately 8,600 tomato genes in resistant plants challenged with X. campestris pv. vesicatoria strains expressing wild-type AvrRxv or a catalytic core AvrRxv mutant. In all, 420 genes were identified as differentially modulated by the expression of a functional AvrRxv, including over 15 functional classes of proteins and a large number of transcription factors and signaling components. Findings of this study allow the development of new hypotheses about the molecular basis of recognition between AvrRxv and the corresponding resistance proteins, and set the stage for the dissection of signaling and cellular responses triggered in tomato plants by this avirulence factor.

Another quarter century of great progress in understanding the biological properties of plant viruses

The last quarter century saw a massive application of new molecular and cell biological, and molecular genetical, techniques in research on plant viruses. Hundreds of complete virus genomic nucleotide sequences were analysed, their constituent genes and control elements identified, and similarities and differences revealed in genome organisation, so justifying the modern taxonomic classification of the viruses. Numerous virus replication systems were described and some viral replicases isolated. Especially good progress was made in understanding cell-to-cell and long-distance virus transport within plants; in cloning dominant and recessive plant genes controlling virus resistances, and identifying the cognate viral avirulence factors; in unraveling mechanisms of virus transmission by invertebrate and plasmodiophorid vectors; and in showing through these advances how viruses utilise and subvert endogenous eukaryotic processes. The discovery of gene silencing, and of viral silencing-suppressor proteins, transformed ideas on how virus replication is controlled, and explained the phenomena of recovery from disease, cross-protection between virus strains and synergy between unrelated viruses. Transgenic, virus-resistant plants were created, tested successfully in field conditions and a few commercialised. Factors underlying the appearance of new disease epidemics were identified. Genetic recombination was reported and found to make an important contribution to generating virus variation, and some major virus evolutionary pathways were recognised by comparative genome analysis. The scene is set for further major advances in the coming decades.

The Pseudomonas syringae type III effector AvrRpt2 promotes virulence independently of RIN4, a predicted virulence target in Arabidopsis thaliana

AvrRpt2, an effector protein from Pseudomonas syringae pv. tomato (Pst), behaves as an avirulence factor that activates resistance in Arabidopsis thaliana lines expressing the resistance gene RPS2. AvrRpt2 can also enhance pathogen fitness by promoting the ability of the bacteria to grow and to cause disease on susceptible lines of A. thaliana that lack functional RPS2. The activation of RPS2 is coupled to the AvrRpt2-induced disappearance of the A. thaliana RIN4 protein. However, the significance of this RIN4 elimination to AvrRpt2 virulence function is unresolved. To clarify our understanding of the contribution of RIN4 disappearance to AvrRpt2 virulence function, we generated new avrRpt2 alleles by random mutagenesis. We show that the ability of six novel AvrRpt2 mutants to induce RIN4 disappearance correlated well with their avirulence activities but not with their virulence activities. Moreover, the virulence activity of wild-type AvrRpt2 was detectable in an A. thaliana line lacking RIN4. Collectively, these results indicate that the virulence activity of AvrRpt2 in A. thaliana is likely to rely on the modification of host susceptibility factors other than, or in addition to, RIN4.

Proteases in pathogenesis and plant defence.

Plant pathogens deliver a variety of virulence factors to host cells to suppress basal defence responses and create suitable environments for their propagation. Plants have in turn evolved disease resistance genes whose products detect the virulence factors as a signal of invasion and activate effective defence responses. Understanding how a virulence effector contributes to virulence on susceptible hosts but becomes an avirulence factor that triggers defence responses on resistance hosts has been a major focus in plant research. Recent studies have shown that a growing list of pathogen-encoded effectors functions as proteases that are secreted into plant cells to modify host proteins. In addition, several plant proteases have been found to function in activation of the defence mechanism. These findings reveal that post-translational modification of host proteins through proteolytic processing is a widely used mechanism in regulating the plant defence response.

Advances in understanding recessive resistance to plant viruses

SUMMARY Recent work carried out to characterize recessive mutations which render experimental hosts non-permissive to viral infection (loss-of-susceptibility mutants) seems to be converging with new data on natural recessive resistance in crop species, and also with functional analyses of virus avirulence determinants. Perhaps the most well known examples are the studies that identified the eukaryotic translation initiation factors 4E(iso) (eIF(iso)4E) and 4E(eIF4E) as the host factors required for potyvirus multiplication within experimental and natural hosts, respectively, and the potyviral genome-linked protein (VPg) as the viral factor that directly interacts with eIF4E to promote potyvirus multiplication. The purpose of this paper is to review the available information on the characterization of loss-of-susceptibility mutants in experimental hosts, natural recessive resistances and virus avirulence factors, and also to comment on possible implications for the design of new sources of sustainable virus resistance.

HRT-mediated Turnip crinkle virus Resistance in Arabidopsis

Turnip crinkle vims (TCV) inoculation onto resistant Arabidopsis ecotype Dijon（Di-17) leads to a hypersensitive response (HR) on the inoculated leaves. A dominant gene, HRT, which confers an HR to TCV, has been cloned from Di-17 plants by map-based cloning. HRT is a LZ-NBS-LRR class resistance gene and it belongs to a small gene family that includes RPP8, which confers resistance to Peronospora parasitica Emco5. Outside of the LRR region, HRT and RPP8 proteins share 98% amino acid identity while their LRR regions are less conserved (87% identity). HRT-transformed Arabidopsis plants developed an HR but generally remained susceptible to TCV due to a dominant RRT allele, which is not compatible with resistance. However, several transgenic plants that over-expressed HRT much higher than Di-l7 showed micro-HR or no HR when inoculated with TCV and were resistant to infection. Both the HR and resistance are dependent on salicylic acid but independent of NPRI, ethylene, or jasmonic acid. Arabidopsis plants containing both TCV coat protein gene and HRT developed massive necrosis and death in seedlings, indicating that the TCV coat protein is an avirulence factor detected by the HRT.

Attenuation of Cf-mediated defense responses at elevated temperatures correlates with a decrease in elicitor-binding sites.

The interaction between the fungal pathogen Cladosporium fulvum and its only host, tomato, is a well-described gene-for-gene system and several resistance (Cf) genes of tomato and matching fungal avirulence (Avr) genes have been characterized. Transgenic tobacco suspension cells expressing Cf genes respond to matching elicitors with typical defense responses, such as medium alkalization and an oxidative burst. We found that this response is attenuated at elevated ambient temperatures. Tomato seedlings expressing both a Cf and the matching Avr gene rapidly die as a result of systemic necrosis at normal temperatures, but are rescued at 33 degrees C. We demonstrate that, at 33 degrees C, the Cf/Avr-mediated induction of defense-related genes is reversibly suppressed. Furthermore, in cell suspensions, the AVR-induced medium alkalization response is slowly suppressed upon incubation at 33 degrees C, but is quickly restored after transfer to lower temperatures. A high-affinity binding site (HABS) for AVR9 is present on plasma membranes isolated from solanaceous plants and has been suggested to act as a co-receptor for AVR9. The amount of AVR9-HABS is 80% reduced in tobacco cell suspensions incubated at 33 degrees C, as compared with cell suspensions incubated at 20 degrees C. Our data suggest that the temperature sensitivity of Cf-mediated defense responses resides at the level of perception of the fungal avirulence factors.

Cutting edge: Salmonella AvrA effector inhibits the key proinflammatory, anti-apoptotic NF-kappa B pathway.

Secreted prokaryotic effector proteins have evolved to modulate the cellular functions of specific eukaryotic hosts. Generally, these proteins are considered virulence factors that facilitate parasitism. However, in certain plant and insect eukaryotic/prokaryotic relationships, effector proteins are involved in the establishment of commensal or symbiotic interactions. In this study, we report that the AvrA protein from Salmonella typhimurium, a common enteropathogen of humans, is an effector molecule that inhibits activation of the key proinflammatory NF-kappaB transcription factor and augments apoptosis in human epithelial cells. This activity is similar but mechanistically distinct from that described for YopJ, an AvrA homolog expressed by the bacterial pathogen Yersinia. We suggest that AvrA may limit virulence in vertebrates in a manner analogous to avirulence factors in plants, and as such, is the first bacterial effector from a mammalian pathogen that has been ascribed such a function.

The Potato virus Y MSNR NIb‐replicase is the elicitor of a veinal necrosis‐hypersensitive response in root knot nematode resistant tobacco

Summary A root knot nematode resistance gene in Nicotiana tabacum, Rk, providing resistance to the nematode parasite Meloidogyne incognita is tightly linked to, or is a pleiotropic gene with a veinal necrosis systemic hypersensitive response to infection by Potato virus Y (PVY) M(s)N(r). The single PVY M(s)N(r) open reading frame was sequenced and found to have 89% protein identity to PVY N. Individual PVY M(s)N(r) polypeptides were deduced and the corresponding cDNA were cloned into a Potato virus X (PVX) based expression vector and used as templates for in vitro transcriptions. Infected plant sap, from N. benthamiana inoculated with infectious RNA, was used to inoculate both root knot nematode (RKN) resistant and susceptible tobacco lines. Lines were then evaluated for the induction of the hypersensitive response. The PVY M(s)N(r) NIb-replicase protein was found to induce a hypersensitive response 10 days post inoculation in nematode resistant tobacco. None of the other PVX/PVY M(s)N(r) constructs induced a hypersensitive response. The NIb-replicase of PVY N, which shares 93% identity to PVY M(s)N(r), did not induce a hypersensitive response when expressed from the PVX vector. This confirmed that the PVY M(s)N(r) NIb-replicase is the elicitor of PVY M(s)N(r) veinal necrosis on RKN plants and thus the first report of a Potyvirus replicase functioning as an avirulence factor.

Host-selective toxins and avirulence determinants: what's in a name?

Host-selective toxins, a group of structurally complex and chemically diverse metabolites produced by plant pathogenic strains of certain fungal species, function as essential determinants of pathogenicity or virulence. Investigations into the molecular and biochemical responses to these disease determinants reveal responses typically associated with host defense and incompatibility induced by avirulence determinants. The characteristic responses that unify these disparate disease phenotypes are numerous, yet the evidence implicating a causal relationship of these responses, whether induced by host-selective toxins or avirulence factors, in determining the consequences of the host-pathogen interaction is equivocal. This review summarizes some examples of the action of host-selective toxins to illustrate the similarity in responses with those to avirulence determinants.

Potentially durable resistance mechanisms in plants to specialised fungal pathogens

Summary Specialised plant pathogens are in many ways adapted to exploit their host plants. Infective propagules should reach the appropriate plant tissue, gain access to the tissue and negate or suppress various kinds of constitutive and inducible resistance mechanisms. The resistance type most frequently deployed in plant breeding is the racespecific resistance, where a hypersensitive response of plant tissue is elicited by an avirulence factor produced by the pathogen. The great disadvantage of this type of resistance is, that it is often ephemeral. Detailed screening of germplasm may result in the discovery of alternative defence mechanisms not associated with hypersensitivity, that may be durable. Avoidance mechanisms may reduce the chance of infection. Upright plant habit has been reported to decrease spore deposition in cereals. Crop architecture may also affect aspects such as humidity and aeration in the crop, and hence the chances for successful infection by pathogen propagules. Other examples of avoidance are leaf surface properties that interfere with leaf wettability, germ tube orientation and finding of stomata to enter the leaf. Stomata in some accessions of Hordeum chilense are excessively covered by cuticular wax that prevent rust fungal germ tubes from perceiving the stomata, resulting in failure of penetration of the pathogen into the leaf. There is evidence that in compatible host species, biotrophic pathogenic fungi induce basic compatibility by suppressing defence mechanisms. Failure of this induction results in abortion of the infection attempt. Several cases of apparently durable resistance are discussed that are based on failure of haustorium formation, and are not associated with hypersensitivity. They may represent cases where the pathogen has problems in establishing basic compatibility.

Plant disease resistance: commonality and novelty in multicellular innate immunity.

Pathogen avirulence genes encode for effector molecules that play a crucial role in the process of pathogen colonization of plant tissue. Successful host defense requires rapid and efficient detection of the pathogen avirulence factors. In the last few years, much progress has been made in delineating the plant molecular sentinels that participate in pathogen identification. Because this ability is genetic information that is 'hard-wired' into the genome, it is called 'innate immunity' and it draws its origins from a phylogenetically ancient form of immunity common to plants and animals. Conservation is shown in many of the functional molecular motifs of innate genes such as the Toll/interleukin 1 receptor domains, nucleotide binding domains and structures that contain leucine rich repeats. Novel plant molecular surveillance domains also include pathogen pattern recognition by coiled-coil domains and specialized kinases. The rapid evolution of plant innate immunity genes is readily detected in their sequence polymorphism, by their massive amplification and appearance in the genome in a clustered organization. By comparative biology of highly diverged innate immunity systems we can enhance our appreciation of the truly basic forces that have shaped its evolution in mutlicellular organisms.

Virus variation in relation to resistance-breaking in plants

In plant viruses, genomic variation caused by mutation is enhanced by recombination, pseudo-recombination and acquisition of extra genomic components. Genomic RNA typically has a high mutation rate, and individual virus isolates consist of swarms of mutants, with the consensus sequence changing in response to selection pressure. Recombination is found among field isolates of some RNA viruses, especially tobraviruses. Among DNA viruses, field isolates of geminiviruses show unexpected nucleotide diversity and frequency of recombination, some of it interspecific. Recombination among whitefly-transmitted geminiviruses (begomoviruses) occurring in the same geographical region contributes to their evolutionary divergence, as a group. Several (perhaps potentially all) viral genes can encode an avirulence factor that elicits resistance controlled by a cognate dominant host gene: such factors include viral coat protein, RNA polymerase, movement protein and proteinase. Some resistance-breaking virus variants have merely a single nonsynonymous nucleotide replacement in their avirulence gene but, with more durable resistances, virulence necessitates two or even multiple replacements. The probability of a resistance-breaking variant appearing and spreading also depends on its biological fitness in the absence of the host resistance gene, and on the type of resistance and number of resistance genes to be overcome. Viruses can have particular difficulty in overcoming strong resistance controlled either by multiple recessive genes or by coat-protein transgenes. Resistance acting at the RNA level is exemplified by post-transcriptional gene silencing induced by transgenic viral sequences. This resistance may be broken by variants with >10% nucleotide non-identity distributed along the sequence. Our ability to identify, and potentially to create, durable virus resistance is increasing steadily.

Genetic engineering of plants to enhance resistance to fungal pathogens—a review of progress and future prospects

Recent applications of techniques in plant molecular biology and biotechnology to the study of host–pathogen interactions have resulted in the identification and cloning of numerous genes involved in the defense responses of plants following pathogen infection. These include: genes that express proteins, peptides, or antimicrobial compounds that are directly toxic to pathogens or that reduce their growth in situ; gene products that directly inhibit pathogen virulence products or enhance plant structural defense genes, that directly or indirectly activate general plant defense responses; and resistance genes involved in the hypersensitive response and in the interactions with avirulence factors. The introduction and expression of these genes, as well as of antimicrobial genes from nonplant sources, in a range of transgenic plant species have shown that the development of fungal pathogens can be significantly reduced. The extent of disease reduction varies with the strategy employed as well as with the characteristics of the fungal pathogen, and disease control has never been complete. Manipulation of salicylic acid, ethylene, and cytokinin levels in transgenic plants have provided some interesting results with regard to enhanced disease tolerance or susceptibility. The complex interactions among the expressed gene product, plant species, and fungal pathogen indicate that the response of transgenic plants cannot be readily predicted. Combinations of defense gene products have shown considerably more promise in reducing disease than single-transgene introductions. The use of tissue-specific or pathogen-inducible promoters, and the engineered expression of resistance genes, synthetic antimicrobial peptides, and elicitor molecules that induce defense responses have the potential to provide commercially useful broad-spectrum disease resistance in the not-too-distant future. The issues and challenges that will need to be addressed prior to the widespread utilization of these transgenic plants are highlighted.

Growth Complementation of hrpXo Mutants of Xanthomonas oryzae pv. oryzae by Virulent Strains in Rice Cultivars Resistant and Susceptible to the Parental Strain

pv. oryzae (X. o. pv. oryzae) T7174 is virulent on rice cultivar IR24 and avirulent on IR-BB2. From recent reports, some virulence and avirulence factors of plant pathogenic bacteria are transferred to plant cells through the hrp-dependent type III secretion system. In this study, we investigated the involvement of hrp genes in the compatible and the incompatible interactions between rice and X. o. pv. oryzae after co-inoculation with hrpXo mutants derived from T7174 and virulent strains. Growth of the mutants, named 74ΔHrpXo and 76ΔHrpXo, was repressed in IR24 when the mutants were applied alone. However, growth of the mutants was complemented by co-inoculation with virulent strains. Growth of bioluminescent hrpXo mutant 76ΔHrpXo in IR24 and its growth in IR-BB2 after co-inoculation with T7133, which is virulent on both cultivars, was equally complemented, as detected by bioluminescence from the mutant. On the other hand, only partial complementation of growth of T7174L76, which is a bioluminescent and pathogenic derivative of T7174, by T7133 was observed in IR-BB2. Thus, growth of the hrpXo mutant of X. o. pv. oryzae was complemented by virulent strains in both susceptible and resistant rice leaves with the parental strain.

Requirement for either a host- or pectin-induced pectate lyase for infection of Pisum sativum by Nectria hematococca.

Fungal pathogens usually have multiple genes that encode extracellular hydrolytic enzymes that may degrade the physical barriers in their hosts during the invasion process. Nectria hematococca, a plant pathogen, has two inducible pectate lyase (PL) genes (pel) encoding PL that can help degrade the carbohydrate barrier in the host. pelA is induced by pectin, whereas pelD is induced only in planta. We show that the disruption of either the pelA or pelD genes alone causes no detectable decrease in virulence. Disruption of both pelA and pelD drastically reduces virulence. Complementation of the double disruptant with pelD gene, or supplementation of the infection droplets of the double disruptant with either purified enzyme, PLA, or PLD, caused a recovery in virulence. These results show that PL is a virulence factor. Thus, we demonstrate that disruption of all functionally redundant genes is required to demonstrate the role of host barrier-degrading enzymes in pathogenesis and that dismissal of the role of such enzymes based on the effects of single-gene disruption may be premature.