Tomato Ve-resistance locus: resilience in the face of adversity?

MAIN CONCLUSION: The Ve-resistance locus in tomato acts as a resilience gene by affecting both the stress/defense cascade and growth, constituting a signaling intercept with a competitive regulatory mechanism. For decades, the tomato Ve-gene has been recognized as a classical resistance R-gene, inherited as a dominant Mendelian trait and encoding a receptor protein that binds with a fungal effector to provide defense against Verticillium dahliae and V. albo-atrum. However, recent molecular studies suggest that the function and role(s) of the Ve-locus and the two proteins that it encodes are more complex than previously understood. This review summarizes both the background and recent molecular evidence and provides a reinterpretation of the function and role(s) of the Ve1- and Ve2-genes and proteins that better accommodates existing data. It is proposed that these two plasma membrane proteins interact to form a signaling intercept that directly links defense and growth. The induction of Ve1 by infection or wounding promotes growth but also downregulates Ve2 signaling, resulting in a decreased biosynthesis of PR proteins. In this context, the Ve1 R-gene acts as a Resilience gene rather than a Resistance gene, promoting taller more robust tomato plants with reduced symptoms (biotic and abiotic) and Verticillium concentration.

The Ve-resistance locus, a plant signaling intercept.

MAIN CONCLUSION: The Ve-resistance locus in tomato and potato affects both stress/defense and growth, consistent with a signaling intercept and a competitive regulatory mechanism. Acting in an antagonistic fashion, the two genes comprising the tomato Ve-resistance locus have been shown to influence both the defense/stress cascade, which causes wilt symptoms, and plant growth (Nazar et al. in Planta 247:1339-1350, 2018c); in contrast, both have been reported to elevate wilt resistance in potato or Arabidopsis. In a further examination of this influence in potato transformed with the Ve1 gene, effects are again demonstrated with respect to both disease resistance and crop productivity consistent with the Ve locus being a signaling intercept and the antagonistic effects, previously observed in tomato. The results support a competitive model in which the tomato Ve1 and Ve2 proteins act to reduce the detrimental effects of the defense/stress cascade and energy transfers to the developing potato tubers.

Wounding induces tomato Ve1 R-gene expression.

MAIN CONCLUSION: In tomato, Ve1 gene expression is induced specifically by physical damage or plant wounding, resulting in a defense/stress cascade that mimics responses during Verticillium colonization and wilt. In tomato, Verticillium resistance is determined by the Ve gene locus, which encodes two leucine-rich repeat-receptor-like proteins (Ve1, Ve2); the Ve1 gene is induced differentially while Ve2 is constitutively expressed throughout disease development. These profiles have been observed even during compatible Verticillium interactions, colonization by some bacterial pathogens, and growth of transgenic tomato plants expressing the fungal Ave1 effector, suggesting broader roles in disease and/or stress. Here, we have examined further Ve gene expression in resistant and susceptible plants under abiotic stress, including a water deficit, salinity and physical damage. Using both quantitative RT-PCR and label-free LC-MS methods, changes have been evaluated at both the mRNA and protein levels. The results indicate that Ve1 gene expression responds specifically to physical damage or plant wounding, resulting in a defense/stress cascade that resembles observations during Verticillium colonization. In addition, the elimination or reduction of Ve1 or Ve2 gene function also result in proteomic responses that occur with wilt pathogen and continue to be consistent with an antagonistic relationship between the two genes. Mutational analyses also indicate the plant wounding hormone, systemin, is not required, while jasmonic acid again appears to play a direct role in induction of the Ve1 gene.

Antagonistic function of the Ve R-genes in tomato.

Key message In Verticillium wilt, gene silencing indicates that tomato Ve2-gene expression can have a dramatic effect on many defense/stress protein levels while Ve1-gene induction modulates these effects in a negative fashion. In tomato, Verticillium resistance is dependent on the Ve R-gene locus, which encodes two leucine-rich repeat receptor-like proteins, Ve1 and Ve2. During fungal wilt, Ve1 protein is sharply induced while Ve2 appears expressed constitutively throughout disease development; the disease resistance function usually is attributed to the Ve1 receptor alone. To study Ve2 function, levels of Ve2 mRNA were suppressed using RNAi in both susceptible and resistant Craigella tomato near-isolines and protein changes were evaluated at both the mRNA and protein levels. The results indicate that Ve2-gene expression can have dramatic effects on many defense/stress protein levels while the presence of intact Ve1 protein minimizes these effects in a negative fashion. The data suggest an antagonistic relationship between the Ve proteins in which Ve1 modulates the induction of defense/stress proteins by Ve2.

A Graft Mimic Strategy for Verticillium Resistance in Tomato.

Grafting vegetables for disease resistance has increased greatly in popularity over the past 10 years. Verticillium wilt of tomato is commonly controlled through grafting of commercial varieties on resistant rootstocks expressing the Ve1 R-gene. To mimic the grafted plant, proteomic analyses in tomato were used to identify a suitable root-specific promoter (TMVi), which was used to express the Ve1-allele in susceptible Craigella (Cs) tomato plants. The results indicate that when infected with Verticillim dahliae, race 1, the transformed plants are comparable to resistant cultivars (Cr) or grafted plants.

Defence cascade in Verticillium-infected grafted tomato.

In tomato the Ve1-gene provides resistance to the vascular pathogen, Verticillium dahliae, race 1; ve1 plants are susceptible. Reciprocal grafts of resistant and susceptible tomato near-isolines were used to examine proteomic changes and, in particular, the effect of the Ve1-gene on the defence/stress protein cascade induced during Verticillium wilt. Based on label-free LC-MS, the results indicate that this defence response is cell-specific, correlates with overall fungal colonization and is mitigated by Ve1 function. The influence of the Ve1-gene in resistant tissues, however, is not actually transferred to susceptible tissues in the grafted plant.

Tomato Ve resistance locus; defense or growth.

MAIN CONCLUSION: Verticillium colonization does induce a cascade of defense/stress proteins but the Ve1 gene also promotes enhanced root growth, which appears to allow the plant to outgrow the pathogen and avoid symptoms associated with an exaggerated defense response. In tomato, the Ve1 gene provides resistance to the vascular pathogen, Verticillium dahliae, race 1; ve1 plants are susceptible. However, the physiological basis of the resistance is unknown. While developing alternative lines of mutant Ve1 gene transformants to address this question a striking difference was observed in transformation frequency resulting from the inefficient rooting of plantlets from ve1 callus relative to Ve1 callus. Subsequent experiments with resistant and susceptible near-isolines of the cultivar Craigella, as well as Ve1 transformants, showed that in both artificial medium and soil, root growth was significantly enhanced in the resistant cultivar. Parallel studies of Verticillium colonization indicated a significantly lower overall concentration in the resistant plant characteristic of the resistant phenotype, but an almost equal total fungal biomass in both resistant and susceptible roots. Proteomic analyses of the roots of Verticillium-infected plants revealed elevated levels of defense/stress proteins, which correlated with the fungal concentration rather than resistance. Hormone analyses demonstrated a higher cis-ABA level in the resistant isoline consistent with enhanced root growth. Taken together these studies indicate a similar fungal biomass in the roots of both isolines where the Ve1 gene also promotes root production. In the case of the Craigella/Vd1 pathosystem, this appears to allow the host to resist better by outgrowing the pathogen with less wilt rather than reliance only on partial immunity.

Biotic factors that induce the tomato Ve1 R-gene.

In tomato, Verticillium resistance is determined by the Ve gene locus encoding two leucine-rich repeat-receptor-like proteins (Ve1, Ve2). The resistance function usually is attributed to Ve1 alone, with two known alleles: Ve1, encoding a resistance protein, and ve1, with a premature stop codon encoding a truncated product. We have examined further Ve-gene expression in resistant and susceptible near-isolines of Verticillium-infected Craigella tomatoes, using both quantitative RT-PCR and an alternative RFLP assay. Ve1 is induced differentially in resistant and susceptible plants, while Ve2 is constitutively expressed throughout disease development. Contrary to their putative role in Verticillium resistance, these profiles were observed even with compatible Verticillium interactions, some bacterial pathogens, and transgenic tomato plants expressing the fungal Ave1 effector. This suggests broader roles in disease and/or stress. To determine the contribution of plant hormones, abscisic acid, methyl jasmonate, naphthaleneacetic acid or salicylic acid were infused independently via the tomato root and effects on Ve1 induction were confirmed using biosynthesis mutants. While all the hormones modulated Ve1-gene induction, abscisic acid and salicylic acid were not required while jasmonic acid appears to play a more direct role.

Verticillium Ave1 effector induces tomato defense gene expression independent of Ve1 protein.

Verticillium resistance is thought to be mediated by Ve1 protein, which presumably follows a "gene-for-gene" relationship with the V. dahliae Ave1 effector. Because in planta analyses of Ave1 have relied so far on transient expression of the gene in tobacco, this study investigated gene function using stably expressing 35S:Ave1 transgenic tomato. Transgenic Ave1 expression was shown to induce various defense genes including those coding for PR-1 (P6), PR-2 (ßbeta-1,3-glucanase) and peroxidases (anionic peroxidase 2, Cevi16 peroxidase). Since a Ve1- tomato cultivar served as germplasm, these results indicate that Ave1 induces these defense genes independently of Ve1.

Arsenal of elevated defense proteins fails to protect tomato against Verticillium dahliae.

Although the hypersensitive reaction in foliar plant diseases has been extensively described, little is clear regarding plant defense strategies in vascular wilt diseases affecting numerous economically important crops and trees. We have examined global genetic responses to Verticillium wilt in tomato (Lycopersicon esculentum Mill.) plants differing in Ve1 resistance alleles. Unexpectedly, mRNA analyses in the susceptible plant (Ve1-) based on the microarrays revealed a very heroic but unsuccessful systemic response involving many known plant defense genes. In contrast, the response is surprisingly low in plants expressing the Ve1+ R-gene and successfully resisting the pathogen. Similarly, whole-cell protein analyses, based on 2D gel electrophoresis and mass spectrometry, demonstrate large systemic increases in a variety of known plant defense proteins in the stems of susceptible plants but only modest changes in the resistant plant. Taken together, the results indicate that the large systemic increases in plant defense proteins do not protect the susceptible plant. Indeed, since a number of the highly elevated proteins are known to participate in the plant hypersensitive response as well as natural senescence, the results suggest that some or all of the disease symptoms, including ultimate plant death, actually may be the result of this exaggerated plant response.

Pac1 endonuclease and Dhp1p 5'-->3' exonuclease are required for U3 snoRNA termination in Schizosaccharomyces pombe.

Maturation of some snoRNAs is dependent on RNase III-like endonuclease-mediated transcript cleavage, which serves as an entry for the nuclear exosome complex that trims the transcript at the 3'-end. Sequence deletions suggest this cleavage in the U3 snoRNA transcripts of Schizosaccharomyces pombe can induce transcript termination. Using mutational analyses, we demonstrate that the degree of cleavage correlates closely with both RNA maturation and transcript termination. We also show that the RNase III-like endonuclease, Pac1, and the nuclear 5'-exonuclease, Dhp1p, are essential for RNA production and transcript termination, supporting a "reversed torpedoes" model in which the endonuclease cut allows 5'- and 3'-exonuclease activities access to the transcript, leading simultaneously to transcript termination in one direction and RNA maturation in the other.

Cleavage-induced termination in U2 snRNA gene expression.

The maturation of many small nuclear RNAs is dependent on RNase III-like endonuclease mediated cleavage, which generates a loading site for the exosome complex that trims the precursor at its 3' end. Using a temperature sensitive Pac1 nuclease, here we show that the endonuclease cleavage is equally important in terminating the transcription of the U2 snRNA in Schizosaccharomyces pombe. Using a temperature sensitive Dhp1p 5'-->3' exonuclease, we demonstrate that it also is an essential component of the termination pathway. Taken together the results support a "reversed torpedoes" model for the termination and maturation of the U2 snRNA; the Pac1 endonuclease cleavage provides entry sites for the 3' and 5' exonuclease activities, leading to RNA maturation in one direction and transcript termination in the other.

DNA chip analysis in diverse organisms with unsequenced genomes.

Whether for basic research or biotechnology, DNA microarrays have become indispensable tools for studying the transcriptome. Normally, analyses begin with a set of known cDNA sequences to prepare microarray chips specific for a target organism with an extensively sequenced and annotated genome. For many organisms, however, genome programs are not complete or have not been initiated. The present study demonstrates that, whether using homologous or heterologous arrays, the chances of seeing interesting differences are similar. When a specific DNA microarray is not available, the results indicate that a reverse approach based on a heterologous array can be used to probe for interesting differences in gene expression. This may be sufficient in many studies but, if necessary, the genes exhibiting the most significant changes subsequently could be identified by traditional molecular approaches. Such a reverse strategy can provide a convenient and inexpensive approach to probe for significant genetic changes in many diverse studies, to monitor or mine critical biological information for basic or applied research, long before complete sequence data are available.

Fail-safe termination elements: a common feature of the eukaryotic genome?

A recent scan of the human genome (1) identified approximately 11 million hairpins. Some have been linked to known sequences, such as viruses, transposable elements, and, more recently, regulating or microRNAs, but the significance, if any, of most sequences that can be predicted to form hairpins remains unknown. Here we show that hairpins that are cleaved by RNase III- like nucleases can induce termination, even with normally polyadenylated transcripts, and that a cleaved hairpin downstream of a normal termination signal can induce fail-safe termination. Because such cleavage sites appear common to intergenic regions, the results raise the possibility that similar fail-safe termination elements are widely distributed in the eukaryotic genome to prevent read-through transcription from disrupting downstream promoter elements or opposing transcription.-Nabavi, S., Nazar, R. N. Fail-safe termination elements: a common feature of the eukaryotic genome?

Endophyte-induced Verticillium protection in tomato is range-restricted.

Endophytes, bacterial, fungal or viral, colonize plants often without causing visible symptoms. More important, they may benefit host plants in many ways, most notably by preventing diseases caused by normally virulent pathogens. Previous studies have shown that an isolate of V. dahliae from eggplant, Dvd-E6, can colonize tomato endophytically, producing taller and more robust tomato plants while providing protection against a virulent V. dahliae, race 1 (Vd1) isolate. Expression analyses suggest this requires interplay between Dvd-E6 and the plant that involves resistance and defense genes. To examine the possibility of a broader effect, dual interactions have been further examined with a more distantly related pathogen, Verticillium albo-atrum (Vaa). The results indicate Dvd-E6 colonization selectively modifies the expression of specific tomato genes to be detrimental to Vd1 but not Vaa, providing evidence that Verticillium-induced protection is range-restricted.

Genetic dissection of Verticillium wilt resistance mediated by tomato Ve1.

Vascular wilt diseases caused by soil-borne pathogens are among the most devastating plant diseases worldwide. The Verticillium genus includes vascular wilt pathogens with a wide host range. Although V. longisporum infects various hosts belonging to the Cruciferaceae, V. dahliae and V. albo-atrum cause vascular wilt diseases in over 200 dicotyledonous species, including economically important crops. A locus responsible for resistance against race 1 strains of V. dahliae and V. albo-atrum has been cloned from tomato (Solanum lycopersicum) only. This locus, known as Ve, comprises two closely linked inversely oriented genes, Ve1 and Ve2, that encode cell surface receptor proteins of the extracellular leucine-rich repeat receptor-like protein class of disease resistance proteins. Here, we show that Ve1, but not Ve2, provides resistance in tomato against race 1 strains of V. dahliae and V. albo-atrum and not against race 2 strains. Using virus-induced gene silencing in tomato, the signaling cascade downstream of Ve1 is shown to require both EDS1 and NDR1. In addition, NRC1, ACIF, MEK2, and SERK3/BAK1 also act as positive regulators of Ve1 in tomato. In conclusion, Ve1-mediated resistance signaling only partially overlaps with signaling mediated by Cf proteins, type members of the receptor-like protein class of resistance proteins.

Plant-endophyte interplay protects tomato against a virulent Verticillium.

Endophytes, bacterial, fungal or viral, colonize plants often without causing visible symptoms. More important, they may benefit host plants in many ways, most notably by preventing diseases caused by normally virulent pathogens. Craigella tomatoes (Lycopersicon esculentum Mill.) can be infected with Verticillium dahliae Kleb., either race 1 (Vd1) or a non-host isolate Dvd-E6 resulting in susceptibility or tolerance, respectively. The present study sought to determine whether Dvd-E6 is endophytic and can protect tomato against Vd1. The total amount of Verticillium in stems and roots was determined by quantitative PCR; the relative amounts of Vd1 and Dvd-E6 were assessed by restriction fragment polymorphism. When Dvd-E6 infects before or together with Vd1, Vd1 is excluded almost completely from the root but, when Vd1 infects first, Dvd-E6 can compete on an equal basis. Previous studies suggested that Dvd-E6 suppresses symptom-related genes, raising the possibility that Dvd-E6 simultaneously induces tolerance to Vd1. This does not seem to be entirely the case since the minimal symptoms following Vd1 infection of Dvd-E6 tolerant Craigella result, at least in part, from restricted Vd1 colonization. Furthermore, when Vd1 and Dvd-E6 are cultured on PDA plates alone or together, the growth rates are similar and neither is inhibitory to the other. Dvd-E6 does not outgrow or inhibit Vd1, in vitro. The protective effect apparently requires interplay between Dvd-E6 and the plant. Expression analyses of tomato genes involved in resistance and defence support this interpretation.

Tomato phenylalanine ammonia-lyase gene family, highly redundant but strongly underutilized.

Phenylalanine ammonia-lyase (PAL) is an important enzyme in both plant development and pathogen defense. In all plants it is encoded by a multi-gene family, ranging in copy number from four in Arabidopsis to a dozen or more copies in some higher plants. Many studies indicate that alternate genes are differentially regulated in response to environmental stimuli. In this study, Southern blot and dot blot analyses in tomato indicate a surprisingly large family of related sequences with approximately 26 copies in the diploid genome, some easily distinguished by restriction enzyme digestion. Analyses of a BAC genome library suggest that the genes are generally not clustered. A more detailed comparison of the gene sequences using PCR to isolate the individual copies and reverse transcription-PCR to study the transcripts that they encode indicates a significant diversity in the gene sequences themselves, but surprisingly only one mRNA transcript can be detected even when additional expression is induced by pathogen growth or wounding. Consistent with previous reports in other plants, a parallel study with a closely related plant, the potato, indicates a much broader utilization of the PAL genes, highlighting the unusual nature of this family in tomato and of the mechanism(s) that silences so many members. Plant transformation analyses further demonstrate the presence of very active silencing, suggesting aggressive competition between PAL gene duplication and copy inactivation during PAL gene evolution.

U3 snoRNA promoter reflects the RNA's function in ribosome biogenesis.

An efficiently expressed "tagged" gene system was used to study promoter sequence elements in genes encoding the U3 snoRNAs of Schizosaccharomyces pombe. Transcription was found dependent on two previously thought, mutually exclusive elements, a TATA-box, as found in other S. pombe snRNA gene promoters, and a Homol D-box, often considered a TATA-box analogue in the promoters of genes that encode ribosomal proteins. The Homol D-box is critical while the TATA-box strongly influences transcription efficiency but is not essential. The results suggest that the U3 snoRNA promoter may represent the fusion of two promoter systems reflecting the special role of this RNA in ribosome biogenesis. Steady state measurements of cellular RNAs support such a coordinated regulation of the U3 snoRNA.

Post-transcriptional regulation of the U3 small nucleolar RNA.

A high copy shuttle vector was used to express a "tagged" U3 small nucleolar RNA (snoRNA) gene in Schizosaccharomyces pombe to examine regulatory responses to a high gene dosage. RNA analyses utilizing reverse transcription-PCR amplification and restriction fragment length polymorphism indicated that the tagged gene was both proportionally and highly expressed and that downstream processing and/or termination were critical to U3 snoRNA stability. In contrast, direct measurements of the total cellular U3 snoRNA showed essentially normal levels of mature RNA, although measurements of precursor levels confirmed a highly expressed gene construct. Taken together, the results indicated that the steady state amounts of mature U3 snoRNA were primarily regulated at the post-transcriptional level. This regulatory mechanism prevents over-accumulation of the cellular U3 snoRNA and can efficiently degrade mutant RNA molecules. Together with past studies on other 3' extended RNA precursors, the results support post-transcriptional regulation as a quality control mechanism in which appropriate amounts of functional RNA are stabilized by protein interaction while excess or defective RNA is rapidly degraded. Precursor processing in vitro and mutational analyses were consistent with this model.