Evaluation of Arabidopsis thaliana as a model host for Xylella fastidiosa

The bacterium Xylella fastidiosa causes a number of plant diseases of significant economic impact. To date, progress determining mechanisms of host-plant susceptibility, tolerance, or resistance has been slow, due in large part to the long generation time and limited available genetic resources for grape, almond, and other known hosts of X. fastidiosa. To overcome many of these limitations, Arabidopsis thaliana has been evaluated as a host for X. fastidiosa. A pin-prick inoculation method has been developed to infect Arabidopsis with X. fastidiosa. Following infection, X. fastidiosa multiplies and can be detected by microscopy, polymerase chain reaction, and isolation. The ecotypes Van-0, LL-0, and Tsu-1 all allow more growth of strain X. fastidiosa Temecula than the reference ecotype Col-0. Affymetrix ATH1 microarray analysis of inoculated vs. noninoculated Tsu-1 reveals gene expression changes that differ greatly from changes seen after infection with apoplast-colonizing bacteria such as Psuedomonas syringae pvs. tomato or syringae. Many genes responsive to oxidative stress are differentially regulated, while classic pathogenesis-related genes are not induced by X. fastidiosa infection.     

Comparative analysis of expressed sequence tags in resistant and susceptible ecotypes of Arabidopsis thaliana infected with cucumber mosaic virus

Arabidopsis thaliana ecotype Columbia (Col-0) is susceptible to the yellow strain of cucumber mosaic virus [CMV(Y)], whereas ecotype C24 is resistant to CMV(Y). Comprehensive analyses of approximately 9,000 expressed sequence tags in ecotypes Col-0 and C24 infected with CMV(Y) suggested that the gene expression patterns in the two ecotypes differed. At 6, 12, 24 and 48 h after CMV(Y) inoculation, the expression of 6, 30, 85 and 788 genes, respectively, had changed in C24, as opposed to 20, 80, 53 and 150 genes in CMV(Y)-infected Col-0. At 12, 24 and 48 h after CMV(Y) inoculation, the abundance of 3, 10 and 55 mRNAs was altered in both ecotypes. However, at 6 h after CMV(Y) inoculation, no genes were co-induced or co-suppressed in both ecotypes. This differential pattern of gene expression between the two ecotypes at an early stage of CMV(Y) infection indicated that the cellular response for resistance may differ from that resulting in susceptibility at the level detectable by the macroarray. According to the expression pattern at various stages of infection, the expression of many genes could be grouped into clusters using cluster analysis. About 100 genes that encode proteins involved in chloroplast function were categorized into clusters 1 and 4, which had a differentially lower expression in CMV(Y)-inoculated C24. The expression of various genes encoding proteins in the endomembrane system belonged to clusters 2 and 4, which were induced in CMV(Y)-inoculated C24 and Col-0 leaves. Characterization of CMV(Y)-altered gene expression in the two ecotypes will contribute to a better understanding of the molecular basis of compatible and incompatible interactions between virus and host plants.     

<Emphasis Type="Italic">Arabidopsis TTR1</Emphasis> causes LRR-dependent lethal systemic necrosis,rather than systemic acquired resistance,to Tobacco ringspot virus

Most Arabidopsis ecotypes display tolerance to the Tobacco ringspot virus (TRSV), but a subset of Arabidopsis ecotypes, including Estland (Est), develop lethal systemic necrosis (LSN), which differs from the localized hypersensitive responses (HRs) or systemic acquired resistance (SAR) characteristic of incompatible reactions. Neither viral replication nor the systemic movement of TRSV was restricted in tolerant or sensitive Arabidopsis ecotypes; therefore, the LSN phenotype shown in the sensitive ecotypes might not be due to viral accumulation. In the present study, we identified the Est TTR1 gene (tolerance to Tobacco ringspot virus 1) encoding a TIR-NBS-LRR protein that controls the ecotype-dependent tolerant/sensitive phenotypes by a map-based cloning method. The tolerant Col-0 ecotype Arabidopsis transformed with the sensitive Est TTR1 allele developed an LSN phenotype upon TRSV infection, suggesting that the Est TTR1 allele is dominant over the tolerant ttr1 allele of Col-0. Multiple sequence alignments of 10 tolerant ecotypes from those of eight sensitive ecotypes showed that 10 LRR amino acid polymorphisms were consistently distributed across the TTR1/ttr1 alleles. Site-directed mutagenesis of these amino acids in the LRR region revealed that two sites, L956S and K1124Q, completely abolished the LSN phenotype. VIGS study revealed that TTR1 is dependent on SGT1, rather than EDS1. The LSN phenotype by TTR1 was shown to be transferred to Nicotiana benthamiana, demonstrating functional conservation of TTR1 across plant families, which are involved in SGT-dependent defense responses, rather than EDS1-dependent signaling pathways.     

Improvement of the movement and host range properties of a plant virus vector through DNA shuffling

Virus expression vectors based on the tobacco mosaic virus (TMV) genome are powerful tools for foreign gene expression in plants. However, the inclusion of increased genetic load in the form of foreign genes limits the speed of systemic plant invasion and host range of these vectors due to reduced replication and movement efficiencies. To improve these properties of TMV vectors, the gene encoding the 30-kDa movement protein was subjected to mutagenesis and DNA shuffling. A vector that expresses the green fluorescent protein was used to allow simple visual discrimination of mutants with enhanced movement phenotypes. An initial round of mutagenesis produced 53 clones with a faster local movement phenotype. Two subsequent rounds of DNA shuffling produced additional clones that showed further increased rates of cell-to-cell movement and degrees of systemic invasion in restrictive hosts. Surprisingly, sequence analysis of the best performing shuffled genes revealed alterations resulting in coding and silent changes in the movement protein gene. Separation of these coding and silent alterations into distinct gene backgrounds revealed that each contributes to improved movement protein function to differing degrees. The resulting vectors demonstrate that the complex activities of the movement protein genes of viruses can be evolved to have improved movement phenotypes, as evidenced by cell-to-cell and systemic invasion. The experiments produced improved vectors that will be of use both for in planta functional screening and for therapeutic protein production and demonstrated the power of shuffling for plant virus vector improvement.     

Identification of a tolerant locus on Arabidopsis thaliana to hypervirulent beet curly top virus CFH strain

The infection of hosts by the geminivirus depends on the interactions between host and viral factors for viral DNA replication, viral gene expression, and the movement of virus throughout the hosts. This work reports that a hypervirulent strain of Beet curly top virus (BCTV) is different in its ability to infect several ecotypes of Arabidopsis thaliana. Symptoms appeared on Arabidopsis ecotypes around 7 to 10 d after inoculation with BCTV-CFH. Symptoms were more severe in ecotype SKKU including severe leaf curling and development of severely deformed and stunted boting compared to Col-O as a lab standard ecotype. One ecotype Cen-O was asymptomatic to BCTV-CFH infection. Studies of viral DNA replication and virus movement in three excised organs of asymptomatic Cen-O demonstrated that BCTV-CFH could replicate viral DNA and move systemically in this ecotype, suggesting that tolerance was due to the blocks of interactions between host and viral factors on symptom development. This asymptomatic phenotype is similar to the mutation of leftward ORFs, especially ORF R2. Genetic analysis of this ecotype Cen-O indicated that tolerance is due to a single recessive locus.     

A TMV-Cg mutant with a truncated coat protein induces cell death resembling the hypersensitive response in Arabidopsis

Tobacco mosaic virus (TMV)-Cg is able to propagate and multiply systemically to high levels in Arabidopsis thaliana ecotype Col-0. In this study, we obtained a Cg mutant, Cgk1, which expresses a coat protein with a truncated carboxyl terminus. Interestingly, Cgk1 induced necrosis that resembled the hypersensitive response and caused more pronounced disease symptoms than wild type Cg in Arabidopsis Col-0 plants. A reactive oxidative burst occurred prior to this necrosis. We found that expression of the pathogenesis-related gene PR-1 was induced by Cgk1 infection, and also by infection with wild type Cg, but only in npr1-2 mutant plants, not in NahG transgenic plants. These results suggested that PR-1 expression is dependent on the salicylic acid signaling pathway, but is independent of NPR1.     

Transgenic expression of pectin methylesterase inhibitors limits tobamovirus spread in tobacco and Arabidopsis

Plant infection by a virus is a complex process influenced by virus‐encoded factors and host components which support replication and movement. Critical factors for a successful tobamovirus infection are the viral movement protein (MP) and the host pectin methylesterase (PME), an important plant counterpart that cooperates with MP to sustain viral spread. The activity of PME is modulated by endogenous protein inhibitors (pectin methylesterase inhibitors, PMEIs). PMEIs are targeted to the extracellular matrix and typically inhibit plant PMEs by forming a specific and stable stoichiometric 1:1 complex. PMEIs counteract the action of plant PMEs and therefore may affect plant susceptibility to virus. To test this hypothesis, we overexpressed genes encoding two well‐characterized PMEIs in tobacco and Arabidopsis plants. Here, we report that, in tobacco plants constitutively expressing a PMEI from Actinidia chinensis (AcPMEI), systemic movement of Tobacco mosaic virus (TMV) is limited and viral symptoms are reduced. A delayed movement of Turnip vein clearing virus (TVCV) and a reduced susceptibility to the virus were also observed in Arabidopsis plants overexpressing AtPMEI‐2. Our results provide evidence that PMEIs are able to limit tobamovirus movement and to reduce plant susceptibility to the virus.     

Pathogenic interactions between variants of cauliflower mosaic virus and Arabidopsis thaliana

Pathogenic interactions between genetic variants of cauliflower mosaic virus (CaMV) and Arabidopsis thaliana were characterized to identify combinations potentially useful in molecular genetic analysis. Infections of a glabrous mutant (gl1) of Arabidopsis ecotype Columbia (Col-0 gl1) by 30 CaMV isolates were assessed by recording symptom character. Thirteen isolates failed to cause symptoms; the remainder induced symptoms that varied between mild and very severe. Some CaMV isolates produced symptoms in Arabidopsis that differed significantly in severity or character from those produced in a standard host Brassica rapa (turnip). A greater variety of symptom types was observed in a single Arabidopsis ecotype infected with a range of CaMV isolates than was found in a range of Arabidopsis ecotypes infected with a single, typical CaMV isolate (Cabb B-JI). One isolate, Bari-1, that was asymptomatic but accumulated virus in Arabidopsis ecotype Col-0 gl1, caused mild symptoms in ecotype Ler gl1. A hybrid virus constructed from CaMV isolates Cabb B-JI and Bari-1 produced symptoms in Arabidopsis variants that were more severe than in either parental isolate. From a screen of EMS-mutagenized Arabidopsis, one mutant (Col-0 dv1) with a pale-green, dark-vein phenotype which had an altered symptom response to CaMV, was isolated. From this, a phenotypically near-normal revertant (Col-0 dv1R) spontaneously arose, but which showed altered responses to CaMV. Infection of Col-0 dv1R by CaMV isolate Bari-1 elicited symptoms unlike the parent Arabidopsis ecotype (Col-0 gl1). Also, Col-0 dv1 and Col-0 dv1R expressed an uncharacteristic necrotic reaction to CaMV.     

Replication of tobacco mosaic virus RNA

The replication of tobacco mosaic virus (TMV) RNA involves synthesis of a negative-strand RNA using the genomic positive-strand RNA as a template, followed by the synthesis of positive-strand RNA on the negative-strand RNA templates. Intermediates of replication isolated from infected cells include completely double-stranded RNA (replicative form) and partly double-stranded and partly single-stranded RNA (replicative intermediate), but it is not known whether these structures are double-stranded or largely single-stranded in vivo. The synthesis of negative strands ceases before that of positive strands, and positive and negative strands may be synthesized by two different polymerases. The genomic-length negative strand also serves as a template for the synthesis of subgenomic mRNAs for the virus movement and coat proteins. Both the virus-encoded 126-kDa protein, which has amino-acid sequence motifs typical of methyltransferases and helicases, and the 183-kDa protein, which has additional motifs characteristic of RNA-dependent RNA polymerases, are required for efficient TMV RNA replication. Purified TMV RNA polymerase also contains a host protein serologically related to the RNA-binding subunit of the yeast translational initiation factor, eIF3. Study of Arabidopsis mutants defective in RNA replication indicates that at least two host proteins are needed for TMV RNA replication. The tomato resistance gene Tm-1 may also encode a mutant form of a host protein component of the TMV replicase. TMV replicase complexes are located on the endoplasmic reticulum in close association with the cytoskeleton in cytoplasmic bodies called viroplasms, which mature to produce 'X bodies'. Viroplasms are sites of both RNA replication and protein synthesis, and may provide compartments in which the various stages of the virus mutiplication cycle (protein synthesis, RNA replication, virus movement, encapsidation) are localized and coordinated. Membranes may also be important for the configuration of the replicase with respect to initiation of RNA synthesis, and synthesis and release of progeny single-stranded RNA.     

Characterization of a new Arabidopsis mutant exhibiting enhanced disease resistance

In many plant-pathogen interactions, resistance is associated with the synthesis and accumulation of salicylic acid (SA) and pathogenesis-related (PR) proteins. At least two general classes of mutants with altered resistance to pathogen attack have been identified in Arabidopsis. One class exhibits increased susceptibility to pathogen infection; the other class exhibits enhanced resistance to pathogens. In an attempt to identify mutations in resistance-associated loci, we screened a population of T-DNA tagged Arabidopsis thaliana ecotype Wassilewskija (Ws) for mutants showing constitutive expression of the PR-1 gene (cep). A mutant was isolated and shown to constitutively express PR-1, PR-2, and PR-5 genes. This constitutive phenotype segregated as a single recessive trait in the Ws genetic background. The mutant also had elevated levels of SA, which are responsible for the cep phenotype. The cep mutant spontaneously formed hypersensitive response (HR)-like lesions on the leaves and cotyledons and also exhibited enhanced resistance to virulent bacterial and fungal pathogens. Genetic analyses of segregating progeny from outcrosses to other ecotypes unexpectedly revealed that alterations in more than one gene condition the constitutive expression of PR genes in the original mutant. One of the mutations, designated cpr20, maps to the lower arm of chromosome 4 and is required for the cep phenotype. Another mutation, which has been termed cpr21, maps to chromosome 1 and is often, but not always, associated with this phenotype. The recessive nature of the cep trait suggests that the CPR20 and CPR21 proteins may act as negative regulators in the disease resistance signal transduction pathway.     

The leucine-rich repeat domain can determine effective interaction between RPS2 and other host factors in arabidopsis RPS2-mediated disease resistance

Like many other plant disease resistance genes, Arabidopsis thaliana RPS2 encodes a product with nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains. This study explored the hypothesized interaction of RPS2 with other host factors that may be required for perception of Pseudomonas syringae pathogens that express avrRpt2 and/or for the subsequent induction of plant defense responses. Crosses between Arabidopsis ecotypes Col-0 (resistant) and Po-1 (susceptible) revealed segregation of more than one gene that controls resistance to P. syringae that express avrRpt2. Many F(2) and F(3) progeny exhibited intermediate resistance phenotypes. In addition to RPS2, at least one additional genetic interval associated with this defense response was identified and mapped using quantitative genetic methods. Further genetic and molecular genetic complementation experiments with cloned RPS2 alleles revealed that the Po-1 allele of RPS2 can function in a Col-0 genetic background, but not in a Po-1 background. The other resistance-determining genes of Po-1 can function, however, as they successfully conferred resistance in combination with the Col-0 allele of RPS2. Domain-swap experiments revealed that in RPS2, a polymorphism at six amino acids in the LRR region is responsible for this allele-specific ability to function with other host factors.     

Identification of Pseudomonas syringae pathogens of Arabidopsis and a bacterial locus determining avirulence on both Arabidopsis and soybean.

To develop a model system for molecular genetic analysis of plant-pathogen interactions, we studied the interaction between Arabidopsis thaliana and the bacterial pathogen Pseudomonas syringae pv tomato (Pst). Pst strains were found to be virulent or avirulent on specific Arabidopsis ecotypes, and single ecotypes were resistant to some Pst strains and susceptible to others. In many plant-pathogen interactions, disease resistance is controlled by the simultaneous presence of single plant resistance genes and single pathogen avirulence genes. Therefore, we tested whether avirulence genes in Pst controlled induction of resistance in Arabidopsis. Cosmids that determine avirulence were isolated from Pst genomic libraries, and the Pst avirulence locus avrRpt2 was defined. This allowed us to construct pathogens that differed only by the presence or absence of a single putative avirulence gene. We found that Arabidopsis ecotype Col-0 was susceptible to Pst strain DC3000 but resistant to the same strain carrying avrRpt2, suggesting that a single locus in Col-0 determines resistance. As a first step toward genetically mapping the postulated resistance locus, an ecotype susceptible to infection by DC3000 carrying avrRpt2 was identified. The avrRpt2 locus from Pst was also moved into virulent strains of the soybean pathogen P. syringae pv glycinea to test whether this locus could determine avirulence on soybean. The resulting strains induced a resistant response in a cultivar-specific manner, suggesting that similar resistance mechanisms may function in Arabidopsis and soybean.     

RCY1, an Arabidopsis thaliana RPP8/HRT family resistance gene,conferring resistance to cucumber mosaic virus requires salicylic acid,ethylene and a novel signal transduction mechanism

The dominant locus, RCY1, in the Arabidopsis thaliana ecotype C24 confers resistance to the yellow strain of cucumber mosaic virus (CMV-Y). The RCY1 locus was mapped to a 150-kb region on chromosome 5. Sequence comparison of this region from C24 and a CMV-Y-susceptible C24 mutant predicts that the RCY1 gene encodes a 104-kDa CC-NBS-LRR-type protein. The RCY1 gene from C24, when expressed in the susceptible ecotype Wassilewskija (Ws), restricted the systemic spread of virus. RCY1 is allelic to the resistance genes RPP8 from the ecotype Landsberg erecta and HRT from the ecotype Dijon-17, which confer resistance to Peronospora parasitica biotype Emco5 and turnip crinkle virus (TCV), respectively. Examination of RCY1 plants defective in salicylic acid (SA), jasmonic acid (JA) and ethylene signaling revealed a requirement for SA and ethylene signaling in mounting a resistance response to CMV-Y. The RCY1 nahG etr1 double mutants exhibited an intermediate level of susceptibility to CMV-Y, compared to the resistant ecotype C24 and the susceptible ecotypes Columbia and Nossen. This suggests that in addition to SA and ethylene, a novel signaling mechanism is associated with the induction of resistance in CMV-Y-infected C24 plants. Moreover, our results suggest that the signaling pathways downstream of the RPP8, HRT, and RCY1 have evolved independently.     

Environmental regulation of stomatal response in the <Emphasis Type="Italic">Arabidopsis</Emphasis> Cvi-0 ecotype

The Arabidopsis Cape Verde Islands (Cvi-0) ecotype is known to differ from other ecotypes with respect to environmental stress responses. We analyzed the stomatal behavior of Cvi-0 plants, in response to environmental signals. We investigated the responses of stomatal conductance and aperture to high [CO₂] in the Cvi-0 and Col-0 ecotypes. Cvi-0 showed constitutively higher stomatal conductance and more stomatal opening than Col-0. Cvi-0 stomata opened in response to light, but the response was slow. Under low humidity, stomatal opening was increased in Cvi-0 compared to Col-0. We then assessed whether low humidity affects endogenous ABA levels in Cvi-0. In response to low humidity, Cvi-0 had much higher ABA levels than Col-0. However, epidermal peels experiments showed that Cvi-0 stomata were insensitive to ABA. Measurements of organic and inorganic ions in Cvi-0 guard cell protoplasts indicated an over-accumulation of osmoregulatory anions (malate and Cl⁻). This irregular anion homeostasis in the guard cells may explain the constitutive stomatal opening phenotypes of the Cvi-0 ecotype, which lacks high [CO₂]-induced and low humidity-induced stomatal closure.     

Differential induction of acquired resistance and PR gene expression in tobacco by virus infection,ethephon treatment,UV light and wounding

Genes for acidic, extracellular and basic, intracellular pathogenesis-related (PR) proteins of tobacco were studied for their response to tobacco mosaic virus (TMV) infection, ethephon treatment, wounding and UV light. The genes encoding the acidic PR proteins (PR-1, PR-2, PR-3, PR-4 and PR-5) responded similarly to the different forms of stress. They appeared to be highly inducible by TMV, moderately inducible by ethephon treatment and UV light and not inducible by wounding. The genes for the basic counterparts of PR-1, PR-2, PR-3 and PR-5 also displayed a common stress response. However, this response was different from that of the acidic PR proteins. Here, the highest induction was obtained upon ethephon treatment, while the other stress conditions resulted in somewhat lower levels of expression. Most genes for acidic PR proteins are systemically induced in the uninfected upper leaves of TMV-infected plants, whereas the genes encoding the basic PR proteins are not. Increased levels of resistance to TMV, comparable to resistance obtained by pre-infection with the virus, were found in UV-irradiated leaves but not in wounded or ethephon-treated leaves. This indicates that the basic PR proteins are not involved in resistance to TMV infection. Tobacco phenylalanine ammonia-lyase genes were not inducible by the various stress conditions. The implications of these findings in relation to the phenomenon of acquired resistance are discussed.