Avirulence Factors Research Articles

Cleavage of PrePL by Lon promotes growth and pathogenesis in Magnaporthe oryzae.

ATP-dependent Lon proteases function in bacterial pathogenesis by regulating the expression of the Type III secretion system; however, little is known about how Lon proteases regulate fungal pathogenesis. We previously investigated Lon-binding proteins involved in fungal pathogenesis that interact with PrePL, the smallest Magnaporthe oryzae Lon-binding protein. Here, we show that Lon cleaves PrePL and produces Pc, an extracellular 11-kDa isoform with catalase and peroxidase activity. The ΔPrePL loss-of-function strain showed stronger sporulation and accelerated disease development, suggesting a temporally specific negative regulatory mechanism controlled by PrePL in disease progression. Neither the truncated Pc, nor the full-length PrePL missing the Lon cleavage site complemented the ΔPrePL phenotype, suggesting that full-length PrePL and Pc both function in fungal development. PrePL targeted to the mitochondria undergoes hydrolysis by Lon to produce Pc, which accumulates in the fungal apoplast. Importantly, recombinant Pc induced plant defence responses and cell death after being infiltrated into selected plant leaves, indicating that it functions as an avirulence factor. This work thus reveals a novel pathogenic factor in the fungal Lon-mediated pathway. Additionally, our results provide new insight into the functions of a full-length protein and its cleaved isoform in fungal pathogenesis.

The Dual Function of the Fungal Toxin Candidalysin during Candida albicans-Macrophage Interaction and Virulence.

The dimorphic fungus Candida albicans is both a harmless commensal organism on mucosal surfaces and an opportunistic pathogen. Under certain predisposing conditions, the fungus can overgrow the mucosal microbiome and cause both superficial and life-threatening systemic infections after gaining access to the bloodstream. As the first line of defense of the innate immune response, infecting C. albicans cells face macrophages, which mediate the clearance of invading fungi by intracellular killing. However, the fungus has evolved sophisticated strategies to counteract macrophage antimicrobial activities and thus evade immune surveillance. The cytolytic peptide toxin, candidalysin, contributes to this fungal defense machinery by damaging immune cell membranes, providing an escape route from the hostile phagosome environment. Nevertheless, candidalysin also induces NLRP3 inflammasome activation, leading to an increased host-protective pro-inflammatory response in mononuclear phagocytes. Therefore, candidalysin facilitates immune evasion by acting as a classical virulence factor but also contributes to an antifungal immune response, serving as an avirulence factor. In this review, we discuss the role of candidalysin during C. albicans infections, focusing on its implications during C. albicans-macrophage interactions.

Production of Melanin from Aspergillus fumigatus as an Avirulence Factor

In this quantitative examination of melanin was detected in 41 isolates of fumigatus. It was found that the significance of the difference in which the production of melanin (p ˂ 0.001) was the range of melanin extracted from fumigatus between (1.2 - 3.1 mg/mL). The isolates AFU1, AFU14, AFU29, AFU30, and AFU41 indicated that the maximum production ranged between (2.9-3.1 mg/mL). 2 hours at 100°C. It has similarities with standard melanin characters, and the same chemical characters of melanin extracted from the hymen of Bjerkandara adusta have been achieved according to a study (14) meaning there is similarity with standard melanin. The overlay of IR spectra of synthetic melanin extracted from fungal cultures showed a high degree of similarity. Purification of melanin from A. fumigatus. Wave numbers shown in 3381, 2927, 1867, 1531, 1404, 1073 and 651 cm-1 The wave range can be traced to the following chemical groups: 3381 cm-1 attributed to OH bonds, 2927 cm-1 to HC or HC = 0 bonds 1404 (C-CH3), and 651 cm -1 to (acyclic) CH2 bonds.

Comparative secretome analysis of Indian wheat leaf rust pathogen Puccinia triticina

The secretome of two races, 77-5 and 106 of wheat leaf rust pathogen Puccinia triticina with known virulence and avirulent trait, respectively were analyzed in this study. The secretome analysis revealed 546 putative secretory proteins (PSPs) present in the race 77-5, and 481 PSPs in race 106. Race-specific PSPs analysis also showed that race 77-5 had higher number of PSPs (19.72%) in comparison to race 106. Various other gene families like pathogenicity and virulence factor were also expanded in higher number in the race 77-5 and few of them having multiple domains which are known for pathogenicity, were exclusively present in this virulent race. The candidate secretory effector proteins (CSEP) analysis also showed that the virulent race contained 8.98% higher CSEP proteins compared to the avirulent race 106. The results suggest that these genes are playing important roles in their respective race-specific manner. Surprisingly the carbohydrate metabolism-related enzymes were found 5.26% higher in the avirulent race in comparison to the virulent one, and few of them have shown race specificity. The in-silico expression analysis of the selected candidate's genes also revealed their role in the pathogenesis process. The three dimensional (3D) structure predictions were performed for few of the candidate genes that were highly expressed. In this comparative secretome analysis, our findings provide a baseline for the characterization of effectors and avirulence factors in different races of P. triticina.

Abstracts of Concurrent Session Presentations at IS-MPMI XVIII Congress.

Plant NLRs (Nucleotide binding, Leucine-rich repeat Receptors) provide resistance to a range of biotrophic pathogens, by recognition of avirulence factors, and proceeded by initiation of downstream immune responses. NLR signaling is transduced through the N-terminal TIR domain, and resistance is often characterized by localized cell death around the site of infection. How plant TIR domains transduce this signal is still unknown, however, like TIR domains from mammalian NLRs, plant TIR domains form homo- and heterotypic dimers, and this dimerization is required for signaling. Recently, a TIR domain from the mammalian TLR adaptor family, SARM1, was shown to possess NADase activity, the first report of a TIR domain displaying enzymatic activity. Given the structural similarities between SARM TIR and plant TIR domains, the requirement of SARM dimerization for NADase activity, and the lack of downstream binding partners identified for plant NLRs, we hypothesize that some plant TIR domains also possess NADase activity. Here we present biochemical and structural evidence that plant TIR domains possess NADase activity, and new insights into how this activity is involved in eliciting HR. NAD+ is cleaved into ADPR and Nicotinamide in vitro by the TIR domains from RUN1 and L6. NADase activity can be increased by addition of macromolecular crowding agents. Mutations to the NADP+ binding site in the RUN1 structure also effect NADase activity.

The type III effector RipB from Ralstonia solanacearum RS1000 acts as a major avirulence factor in Nicotiana benthamiana and other Nicotiana species.

Summary Ralstonia solanacearum is the causal agent of bacterial wilt in solanaceous crops. This pathogen injects approximately 70 effector proteins into plant cells via the Hrp type III secretion system in an early stage of infection. To identify an as‐yet‐unidentified avirulence factor possessed by the Japanese tobacco‐avirulent strain RS1000, we transiently expressed RS1000 effectors in Nicotiana benthamiana leaves and monitored their ability to induce effector‐triggered immunity (ETI). The expression of RipB strongly induced the production of reactive oxygen species and the expressions of defence‐related genes in N. benthamiana. The ripB mutant of RS1002, a nalixidic acid‐resistant derivative of RS1000, caused wilting symptoms in N. benthamiana. A pathogenicity test using R. solanacearum mutants revealed that the two already known avirulence factors RipP1 and RipAA contribute in part to the avirulence of RS1002 in N. benthamiana. The Japanese tobacco‐virulent strain BK1002 contains mutations in ripB and expresses a C‐terminal‐truncated RipB that lost the ability to induce ETI in N. benthamiana, indicating a fine‐tuning of the pathogen effector repertoire to evade plant recognition. RipB shares homology with Xanthomonas XopQ, which is recognized by the resistance protein Roq1. The RipB‐induced resistance against R. solanacearum was abolished in Roq1‐silenced plants. These findings indicate that RipB acts as a major avirulence factor in N. benthamiana and that Roq1 is involved in the recognition of RipB.

Manipulating Cellular Factors to Combat Viruses: A Case Study From the Plant Eukaryotic Translation Initiation Factors eIF4.

Genes conferring resistance to plant viruses fall in two categories; the dominant genes that mostly code for proteins with a nucleotide binding site and leucine rich repeats (NBS-LRR), and that directly or indirectly, recognize viral avirulence factors (Avr), and the recessive genes. The latter provide a so-called recessive resistance. They represent roughly half of the known resistance genes and are alleles of genes that play an important role in the virus life cycle. Conversely, all cellular genes critical for the viral infection virtually represent recessive resistance genes. Based on the well-documented case of recessive resistance mediated by eukaryotic translation initiation factors of the 4E/4G family, this review is intended to summarize the possible approaches to control viruses via their host interactors. Classically, resistant crops have been developed through introgression of natural variants of the susceptibility factor from compatible relatives or by random mutagenesis and screening. Transgenic methods have also been applied to engineer improved crops by overexpressing the translation factor either in its natural form or after directed mutagenesis. More recently, innovative approaches like silencing or genome editing have proven their great potential in model and crop plants. The advantages and limits of these different strategies are discussed. This example illustrates the need to identify and characterize more host factors involved in virus multiplication and to assess their application potential in the control of viral diseases.

Non-Structural Protein NSm of Tomato Spotted Wilt Virus Is an Avirulence Factor Recognized by Resistance Genes of Tobacco and Tomato via Different Elicitor Active Sites.

Tomato spotted wilt virus (TSWV) is one of the most destructive viral pathogens of plants. Recently, a single dominant gene conferring complete resistance to TSWV (RTSW) was identified in Nicotina alata and introgressed into cultivated tobacco (N. tabacum). However, whether the TSWV carries an avirulence (Avr) factor directed against RTSW remains obscure. In the present study, we identified the non-structural protein (NSm), the movement protein of TSWV, which is an RTSW-specific Avr factor, by using two different transient expression systems. Using amino acid (aa) substitution mutants, we demonstrated the ability to induce RTSW-mediated hypersensitive response (HR) of NSm is independent of its movement function. Moreover, key substitutions (C118Y and T120N), a 21-aa viral effector epitope, and different truncated versions of NSm, which are responsible for the recognition of the Sw-5b resistance gene of tomato, were tested for their ability to trigger HR to TSWV in tobacco. Together, our results demonstrated that RTSW-mediated resistance is triggered by NSm in the same way as by Sw-5b, however, via different elicitor active sites. Finally, an Avr gene-based diagnostic approach was established and used to determine the presence and effectiveness of resistance genes in tobacco.

Fusarium oxysporumcolonizes the stem of resistant tomato plants, the extent varying with the R-gene present

The plant immune system employs resistance (R) genes to detect the presence of pathogenic microbes by the avirulence (Avr) factors they produce. Whereas some R-genes confer extreme resistance, completely blocking pathogen proliferation, others act later during infection and allow initial microbial multiplication in the host. We hypothesized that transmembrane R-proteins – as opposed to intracellular R-proteins - may recognize and arrest pathogen entry earlier in the infection process. In tomato, two transmembrane R-proteins (I and I-3) and one intracellular R-protein (I-2) have been identified conferring resistance against the same pathogen: the soil borne fungus Fusarium oxysporum f.sp. lycopersici (Fol). We found that in all root inoculations with Fol on resistant tomato plants, the fungus was able to reach the vasculature of the stem. However, the extent of host vasculature colonization was less than that in susceptible plants. This indicates that a complete blockade of fungal ingress does not occur in any of the incompatible interactions. However, resistance mediated by the intracellular R-protein I-2 allowed more extensive fungal colonization than resistance mediated by the transmembrane R-proteins I or I-3. The first phases of invasion – penetration of the root epidermis, cortex colonization and early stage xylem colonization – are unaffected by R-gene mediated resistance of the host. We suggest that all 3 R-proteins only limit Fol colonization after the fungus has reached the xylem tissues, and that I and I-3 act prior to and/or are more effective than I-2 in reducing pathogen proliferation and spread.

The Globodera pallida SPRYSEC Effector GpSPRY-414-2 That Suppresses Plant Defenses Targets a Regulatory Component of the Dynamic Microtubule Network.

The white potato cyst nematode, Globodera pallida, is an obligate biotrophic pathogen of a limited number of Solanaceous plants. Like other plant pathogens, G. pallida deploys effectors into its host that manipulate the plant to the benefit of the nematode. Genome analysis has led to the identification of large numbers of candidate effectors from this nematode, including the cyst nematode-specific SPRYSEC proteins. These are a secreted subset of a hugely expanded gene family encoding SPRY domain-containing proteins, many of which remain to be characterized. We investigated the function of one of these SPRYSEC effector candidates, GpSPRY-414-2. Expression of the gene encoding GpSPRY-414-2 is restricted to the dorsal pharyngeal gland cell and reducing its expression in G. pallida infective second stage juveniles using RNA interference causes a reduction in parasitic success on potato. Transient expression assays in Nicotiana benthamiana indicated that GpSPRY-414-2 disrupts plant defenses. It specifically suppresses effector-triggered immunity (ETI) induced by co-expression of the Gpa2 resistance gene and its cognate avirulence factor RBP-1. It also causes a reduction in the production of reactive oxygen species triggered by exposure of plants to the bacterial flagellin epitope flg22. Yeast two-hybrid screening identified a potato cytoplasmic linker protein (CLIP)-associated protein (StCLASP) as a host target of GpSPRY-414-2. The two proteins co-localize in planta at the microtubules. CLASPs are members of a conserved class of microtubule-associated proteins that contribute to microtubule stability and growth. However, disruption of the microtubule network does not prevent suppression of ETI by GpSPRY-414-2 nor the interaction of the effector with its host target. Besides, GpSPRY-414-2 stabilizes its target while effector dimerization and the formation of high molecular weight protein complexes including GpSPRY-414-2 are prompted in the presence of the StCLASP. These data indicate that the nematode effector GpSPRY-414-2 targets the microtubules to facilitate infection.

A fungal avirulence factor encoded in a highly plastic genomic region triggers partial resistance to septoria tritici blotch.

Summary Cultivar‐strain specificity in the wheat–Zymoseptoria tritici pathosystem determines the infection outcome and is controlled by resistance genes on the host side, many of which have been identified. On the pathogen side, however, the molecular determinants of specificity remain largely unknown.We used genetic mapping, targeted gene disruption and allele swapping to characterise the recognition of the new avirulence factor Avr3D1. We then combined population genetic and comparative genomic analyses to characterise the evolutionary trajectory of Avr3D1.Avr3D1 is specifically recognised by wheat cultivars harbouring the Stb7 resistance gene, triggering a strong defence response without preventing pathogen infection and reproduction. Avr3D1 resides in a cluster of putative effector genes located in a genome region populated by independent transposable element insertions. The gene was present in all 132 investigated strains and is highly polymorphic, with 30 different protein variants identified. We demonstrated that specific amino acid substitutions in Avr3D1 led to evasion of recognition.These results demonstrate that quantitative resistance and gene‐for‐gene interactions are not mutually exclusive. Localising avirulence genes in highly plastic genomic regions probably facilitates accelerated evolution that enables escape from recognition by resistance proteins.

Membrane Vesicles Derived from Bordetella bronchiseptica: Active Constituent of a New Vaccine against Infections Caused by This Pathogen.

Bordetella bronchiseptica, a Gram-negative bacterium, causes chronic respiratory tract infections in a wide variety of mammalian hosts, including humans (albeit rarely). We recently designed Bordetella pertussis and Bordetella parapertussis experimental vaccines based on outer membrane vesicles (OMVs) derived from each pathogen, and we obtained protection against the respective infections in mice. Here, we demonstrated that OMVs derived from virulent-phase B. bronchiseptica (OMVBbvir+) protected mice against sublethal infections with different B. bronchiseptica strains, two isolated from farm animals and one isolated from a human patient. In all infections, we observed that the B. bronchiseptica loads were significantly reduced in the lungs of vaccinated animals; the lung-recovered CFU were decreased by ≥4 log units, compared with those detected in the lungs of nonimmunized animals (P < 0.001). In the OMVBbvir+-immunized mice, we detected IgG antibody titers against B. bronchiseptica whole-cell lysates, along with an immune serum having bacterial killing activity that both recognized B. bronchiseptica lipopolysaccharides and polypeptides such as GroEL and outer membrane protein C (OMPc) and demonstrated an essential protective capacity against B. bronchiseptica infection, as detected by passive in vivo transfer experiments. Stimulation of cultured splenocytes from immunized mice with OMVBbvir+ resulted in interleukin 5 (IL-5), gamma interferon (IFN-γ), and IL-17 production, indicating that the vesicles induced mixed Th2, Th1, and Th17 T-cell immune responses. We detected, by adoptive transfer assays, that spleen cells from OMVBbvir+-immunized mice also contributed to the observed protection against B. bronchiseptica infection. OMVs from avirulent-phase B. bronchiseptica and the resulting induced immune sera were also able to protect mice against B. bronchiseptica infection.IMPORTANCEBordetella bronchiseptica, a Gram-negative bacterium, causes chronic respiratory tract infections in a wide variety of mammalian hosts, including humans (albeit rarely). Several vaccines aimed at preventing B. bronchiseptica infection have been developed and used, but a safe effective vaccine is still needed. The significance and relevance of our research lie in the characterization of the OMVs derived from B. bronchiseptica as the source of a new experimental vaccine. We demonstrated here that our formulation based on OMVs derived from virulent-phase B. bronchiseptica (OMVBbvir+) was effective against infections caused by B. bronchiseptica isolates obtained from different hosts (farm animals and a human patient). In vitro and in vivo characterization of humoral and cellular immune responses induced by the OMVBbvir+ vaccine enabled a better understanding of the mechanism of protection necessary to control B. bronchiseptica infection. Here we also demonstrated that OMVs derived from B. bronchiseptica in the avirulent phase and the corresponding induced humoral immune response were able to protect mice from B. bronchiseptica infection. This realization provides the basis for the development of novel vaccines not only against the acute stages of the disease but also against stages of the disease or the infectious cycle in which avirulence factors could play a role.

The TNL gene Rdr1 confers broad-spectrum resistance to Diplocarpon rosae.

Black spot disease, which is caused by the ascomycete Diplocarpon rosae, is the most severe disease in field-grown roses in temperate regions and has been distributed worldwide, probably together with commercial cultivars. Here, we present data indicating that muRdr1A is the active Rdr1 gene, a single-dominant TIR-NBS-LRR (Toll/interleukin-1 receptor-nucleotide binding site-leucine rich repeat) (TNL)-type resistance gene against black spot disease, which acts against a broad range of pathogenic isolates independent of the genetic background of the host genotype. Molecular analyses revealed that, compared with the original donor genotype, the multiple integrations that are found in the primary transgenic clone segregate into different integration patterns in its sexual progeny and do not show any sign of overexpression. Rdr1 provides resistance to 13 different single-spore isolates belonging to six different races and broad field mixtures of conidia; thus far, Rdr1 is only overcome by two races. The expression of muRdr1A, the active Rdr1 gene, leads to interaction patterns that are identical in the transgenic clones and the non-transgenic original donor genotype. This finding indicates that the interacting avirulence (Avr) factor on the pathogen side must be widespread among the pathogen populations and may have a central function in the rose-black spot interaction. Therefore, the Rdr1 gene, pyramided with only a few other R genes by sexual crosses, might be useful for breeding roses that are resistant to black spot because the spread of new pathogenic races of the fungus appears to be slow.

The effector AvrRxo1 phosphorylates NAD in planta.

Gram-negative bacterial pathogens of plants and animals employ type III secreted effectors to suppress innate immunity. Most characterized effectors work through modification of host proteins or transcriptional regulators, although a few are known to modify small molecule targets. The Xanthomonas type III secreted avirulence factor AvrRxo1 is a structural homolog of the zeta toxin family of sugar-nucleotide kinases that suppresses bacterial growth. AvrRxo1 was recently reported to phosphorylate the central metabolite and signaling molecule NAD in vitro, suggesting that the effector might enhance bacterial virulence on plants through manipulation of primary metabolic pathways. In this study, we determine that AvrRxo1 phosphorylates NAD in planta, and that its kinase catalytic sites are necessary for its toxic and resistance-triggering phenotypes. A global metabolomics approach was used to independently identify 3’-NADP as the sole detectable product of AvrRxo1 expression in yeast and bacteria, and NAD kinase activity was confirmed in vitro. 3’-NADP accumulated upon transient expression of AvrRxo1 in Nicotiana benthamiana and in rice leaves infected with avrRxo1-expressing strains of X. oryzae. Mutation of the catalytic aspartic acid residue D193 abolished AvrRxo1 kinase activity and several phenotypes of AvrRxo1, including toxicity in yeast, bacteria, and plants, suppression of the flg22-triggered ROS burst, and ability to trigger an R gene-mediated hypersensitive response. A mutation in the Walker A ATP-binding motif abolished the toxicity of AvrRxo1, but did not abolish the 3’-NADP production, virulence enhancement, ROS suppression, or HR-triggering phenotypes of AvrRxo1. These results demonstrate that a type III effector targets the central metabolite and redox carrier NAD in planta, and that this catalytic activity is required for toxicity and suppression of the ROS burst.

The 50 distal amino acids of the 2AHP homing protein of Grapevine fanleaf virus elicit a hypersensitive reaction on Nicotiana occidentalis.

Avirulence factors are critical for the arm's race between a virus and its host in determining incompatible reactions. The response of plants to viruses from the genus Nepovirus in the family Secoviridae, including Grapevine fanleaf virus (GFLV), is well characterized, although the nature and characteristics of the viral avirulence factor remain elusive. By using infectious clones of GFLV strains F13 and GHu in a reverse genetics approach with wild-type, assortant and chimeric viruses, the determinant of necrotic lesions caused by GFLV-F13 on inoculated leaves of Nicotiana occidentalis was mapped to the RNA2-encoded protein 2AHP , particularly to its 50 C-terminal amino acids. The necrotic response showed hallmark characteristics of a genuine hypersensitive reaction, such as the accumulation of phytoalexins, reactive oxygen species, pathogenesis-related protein 1c and hypersensitivity-related (hsr) 203J transcripts. Transient expression of the GFLV-F13 protein 2AHP fused to an enhanced green fluorescent protein (EGFP) tag in N.occidentalis by agroinfiltration was sufficient to elicit a hypersensitive reaction. In addition, the GFLV-F13 avirulence factor, when introduced in GFLV-GHu, which causes a compatible reaction on N.occidentalis, elicited necrosis and partially restricted the virus. This is the first identification of a nepovirus avirulence factor that is responsible for a hypersensitive reaction in both the context of virus infection and transient expression.

Immune Receptors and Co-receptors in Antiviral Innate Immunity in Plants.

Plants respond to pathogens using an innate immune system that is broadly divided into PTI (pathogen-associated molecular pattern- or PAMP-triggered immunity) and ETI (effector-triggered immunity). PTI is activated upon perception of PAMPs, conserved motifs derived from pathogens, by surface membrane-anchored pattern recognition receptors (PRRs). To overcome this first line of defense, pathogens release into plant cells effectors that inhibit PTI and activate effector-triggered susceptibility (ETS). Counteracting this virulence strategy, plant cells synthesize intracellular resistance (R) proteins, which specifically recognize pathogen effectors or avirulence (Avr) factors and activate ETI. These coevolving pathogen virulence strategies and plant resistance mechanisms illustrate evolutionary arms race between pathogen and host, which is integrated into the zigzag model of plant innate immunity. Although antiviral immune concepts have been initially excluded from the zigzag model, recent studies have provided several lines of evidence substantiating the notion that plants deploy the innate immune system to fight viruses in a manner similar to that used for non-viral pathogens. First, most R proteins against viruses so far characterized share structural similarity with antibacterial and antifungal R gene products and elicit typical ETI-based immune responses. Second, virus-derived PAMPs may activate PTI-like responses through immune co-receptors of plant PTI. Finally, and even more compelling, a viral Avr factor that triggers ETI in resistant genotypes has recently been shown to act as a suppressor of PTI, integrating plant viruses into the co-evolutionary model of host-pathogen interactions, the zigzag model. In this review, we summarize these important progresses, focusing on the potential significance of antiviral immune receptors and co-receptors in plant antiviral innate immunity. In light of the innate immune system, we also discuss a newly uncovered layer of antiviral defense that is specific to plant DNA viruses and relies on transmembrane receptor-mediated translational suppression for defense.

Uptake of the Fusarium Effector Avr2 by Tomato Is Not a Cell Autonomous Event.

Pathogens secrete effector proteins to manipulate the host for their own proliferation. Currently it is unclear whether the uptake of effector proteins from extracellular spaces is a host autonomous process. We study this process using the Avr2 effector protein from Fusarium oxysporum f. sp. lycopersici (Fol). Avr2 is an important virulence factor that is secreted into the xylem sap of tomato following infection. Besides that, it is also an avirulence factor triggering immune responses in plants carrying the I-2 resistance gene. Recognition of Avr2 by I-2 occurs inside the plant nucleus. Here, we show that pathogenicity of an Avr2 knockout Fusarium (FolΔAvr2) strain is fully complemented on transgenic tomato lines that express either a secreted (Avr2) or cytosolic Avr2 (ΔspAvr2) protein, indicating that Avr2 exerts its virulence functions inside the host cells. Furthermore, our data imply that secreted Avr2 is taken up from the extracellular spaces in the presence of the fungus. Grafting studies were performed in which scions of I-2 tomato plants were grafted onto either a ΔspAvr2 or on an Avr2 rootstock. Although the Avr2 protein could readily be detected in the xylem sap of the grafted plant tissues, no I-2-mediated immune responses were induced suggesting that I-2-expressing tomato cells cannot autonomously take up the effector protein from the xylem sap. Additionally, ΔspAvr2 and Avr2 plants were crossed with I-2 plants. Whereas ΔspAvr2/I-2 F1 plants showed a constitutive immune response, immunity was not triggered in the Avr2/I-2 plants confirming that Avr2 is not autonomously taken up from the extracellular spaces to trigger I-2. Intriguingly, infiltration of Agrobacterium tumefaciens in leaves of Avr2/I-2 plants triggered I-2 mediated cell death, which indicates that Agrobacterium triggers effector uptake. To test whether, besides Fol, effector uptake could also be induced by other fungal pathogens the ΔspAvr2 and Avr2 transgenic lines were inoculated with Verticillium dahliae. Whereas ΔspAvr2 plants became hyper-susceptible to infection, no difference in disease development was found in the Avr2 plants as compared to wild-type plants. These data suggest that effector uptake is not a host autonomous process and that Fol and A. tumefaciens, but not V. dahliae, facilitate Avr2 uptake by tomato cells from extracellular spaces.

INVESTIGATION ON TOMATO SPOTTED WILT VIRUS INFECTING PEPPER PLANTS IN HUNGARY

In Hungary resurgence of Tomato spotted wilt virus (TSWV) frequently causesheavy crop losses in pepper production since the mid nineties. Management ofTSWV control was first directed against the thrips (using different insecticides orplastic traps), and against weeds as host plants of the virus and the thrips. Later onTsw resistance gene was introduced from Capsicum chinense PI 152225 and PI159236 into different types of pepper. In 2010 and 2011 sporadically, but in 2012more frequently a resistance breaking (RB) strain of TSWV on resistant peppercultivars was observed in the Szentes region (South-East Hungary). The presenceof a new resistance breaking strain was demonstrated by virological (test-plant,serological and RT-PCR) methods. Previously, the non-structural protein (NSs)encoded by small RNA (S RNA) of TSWV was verified as the avirulence factor forTsw resistance, therefore we analyzed the S RNA of the Hungarian RB and wildtype (WT) isolates and compared to previously analyzed TSWV strains with RBproperties from different geographical origins. Phylogenetic analysis demonstratedthat the different RB strains had the closest relationship with the local WT isolatesand there was no conserved mutation present in all the NSs genes of RB isolatesfrom different geographical origins. According to these results, it is concluded thatthe RB isolates evolved separately in geographic point of view and according to theRB mechanism. In order to find new genetic sources of resistance in Capsicumspecies 89 lines from Capsicum annuum, C. chinense, C. frutescens, C. chacoense,C. baccatum var. baccatum, C. baccatum var. pendulum and C. praetermissumwere tested with the Hungarian TSWV-RB isolate.

Knocking down expression of the auxin-amidohydrolase IAR3 alters defense responses in Solanaceae family plants

In plants, indole-3-acetic acid (IAA) amido hydrolases (AHs) participate in auxin homeostasis by releasing free IAA from IAA-amino acid conjugates. We investigated the role of IAR3, a member of the IAA amido hydrolase family, in the response of Solanaceous plants challenged by biotrophic and hemi-biotrophic pathogens. By means of genome inspection and phylogenic analysis we firstly identified IAA-AH sequences and putative IAR3 orthologs in Nicotiana benthamiana, tomato and potato. We evaluated the involvement of IAR3 genes in defense responses by using virus-induced gene silencing. We observed that N. benthamiana and tomato plants with knocked-down expression of IAR3 genes contained lower levels of free IAA and presented altered responses to pathogen attack, including enhanced basal defenses and higher tolerance to infection in susceptible plants. We showed that IAR3 genes are consistently up-regulated in N. benthamiana and tomato upon inoculation with Phytophthora infestans and Cladosporium fulvum respectively. However, IAR3 expression decreased significantly when hypersensitive response was triggered in transgenic tomato plants coexpressing the Cf-4 resistance gene and the avirulence factor Avr4. Altogether, our results indicate that changes in IAR3 expression lead to alteration in auxin homeostasis that ultimately affects plant defense responses.

The FonSIX6 gene acts as an avirulence effector in the Fusarium oxysporum f. sp. niveum - watermelon pathosystem.

When infecting a host plant, the fungus Fusarium oxysporum secretes several effector proteins into the xylem tissue to promote virulence. However, in a host plant with an innate immune system involving analogous resistance proteins, the fungus effector proteins may trigger resistance, rather than promoting virulence. Identity of the effector genes of Fusarium oxysporum f. sp. niveum (Fon) races that affect watermelon (Citrullus lanatus) are currently unknown. In this study, the SIX6 (secreted in xylem protein 6) gene was identified in Fon races 0 and 1 but not in the more virulent Fon race 2. Disrupting the FonSIX6 gene in Fon race 1 did not affect the sporulation or growth rate of the fungus but significantly enhanced Fon virulence in watermelon, suggesting that the mutant ΔFon1SIX6 protein allowed evasion of R protein-mediated host resistance. Complementation of the wild-type race 2 (which lacks FonSIX6) with FonSIX6 reduced its virulence. These results provide evidence supporting the hypothesis that FonSIX6 is an avirulence gene. The identification of FonSix6 as an avirulence factor may be a first step in understanding the mechanisms of Fon virulence and resistance in watermelon and further elucidating the role of Six6 in Fusarium-plant interactions.