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.Key words: cross-protection, endophyte, lycopersicon, tolerance, VerticilliumAn “endophyte” commonly is defined as a “fungus or bacterium living within plants without causing visible symptoms of disease”; a “pathogen” is regarded as “a disease causing biological agent”. Historically, plant biologists have tended to consider these as two very different and distinct classes of organisms but accumulating evidence now suggests that the boundaries between mutualism and parasitism are not as defined as previously thought. Many organisms can occupy both ecological niches¹ depending on the genotype of the host, the genotype of the organism itself and interaction with the environment. Indeed, this “dual life style” may be a significant factor in the evolutionary dynamics of pathogen resistance, tolerance and susceptibility.²One of the more recent additions to the growing list of dual life style (endophyte/pathogen) fungi is Verticillium dahliae,³ a causal agent of vascular wilt disease or early dying syndrome in a broad range of the plant species.⁴ When Verticillium infects a plant three different host responses can occur: resistance, susceptibility or tolerance. The phenomenon of tolerance has been associated with Verticillium spp. for decades but research on mechanisms governing the development of the plant/Verticillium interaction has focused on the compatible and incompatible interactions and little is known about the tolerant state (reviewed in ref. ⁵). In a recent series of papers we have identified an isolate of Verticillium dahliae, known as Dvd-E6, that colonizes tomatoes, cv Craigella, resulting in a stable tolerant condition, that we have used as a model system to investigate the biological and molecular bases of plant tolerance to Verticillium spp. The Craigella/Dvd E6 interaction has a number of interesting but unanticipated properties. Host plants tend to be taller and more robust than their uninfected counterparts⁶ and colonization of Craigella by Dvd-E6 provides protection against its virulent Verticillium dahliae race 1 (Vd1) cousin, limiting both Vd1 colonization and symptom development during dual infections.⁷ Such attributes normally are associated with plant/endophyte partnerships, strongly suggesting that under some conditions, Verticillium dahliae can assume an endophytic role. Apparently interplay between Dvd-E6 and the plant establishes protection against the virulent Vd1.⁷Many endophytic infections provide protection for the host against a broad range of pathogens.¹ To test the ability of Dvd-E6 infection to protect Craigella against a more distantly related Verticillium pathogen we have now examined dual interactions with Verticillium albo-atrum (Vaa). Susceptible Craigella⁸ seedlings again were inoculated at the 4-leaf stage by dipping the roots in Vd1, Dvd-E6 or Vaa conidial suspensions (1 × 10⁷ spores/ml in 0.5% gelatin solution) to establish homogeneous interactions (reviewed in ref. ⁷). For dual interactions, seedlings were inoculated with Dvd-E6 spores at the 3-leaf stage and reinoculated at the 4-leaf stage with either Vd1 or Vaa spore suspension to establish the mixed infections. Control seedlings were root dipped in gelatin solution alone. Plants were scored for symptom expression on a 0 (no symptoms) to 5 (plant dead) scale as described by Shittu and co-workers (2009) and the top two-thirds of the stems harvested at 5 and 10 days post inoculation (d.p.i.) for extraction and fungal DNA assays.^7,9All experimental results are summarized in Figure 1. At 10 d.p.i., the disease scores for Dvd-E6-infected plants were approximately one-third that of the Vd1-or Vaa-infected plants. More interesting were the symptoms in the dual interactions, Dvd-E6/Vd1 mimicking the tolerant plants with low disease and Dvd-E6/Vaa-infected plants exhibiting the highest disease scores, similar to Vd1-or Vaa-infected plants. When the amount of fungal DNA in the stems was assessed, the total fungal biomass in both dual infections as well as the Dvd-E6 and Vd1-infected plants at 5 and 10 dpi was similar (light and dark gray bars), the amount of Vaa being somewhat lower. In the mixed infections, however, most of the DNA (>90%) was of Dvd-E6 origin (white bars). More important, the Vd1 DNA level in Dvd-E6/Vd1 plants was substantially reduced relative to plants infected with Vd1 alone while the Vaa DNA levels stayed about the same in the single and double infections. This indicates that Vd1 colonization is restricted⁷ by the presence of Dvd-E6 while Vaa is not.Open in a separate windowFigure 1Comparison of symptoms and levels of V. dahliae 1 or V. albo-atrum DNA in susceptible Craigella tomato simultaneously infected with V. dahliae Dvd-E6. Individual (E6, Vd1 and Vaa) and mixed (E6/Vd1 and E6/Vaa) infections were established as previously described.⁷ Plants were scored (upper) at 5 (light gray bars) and 10 (dark gray bars) dpi for symptoms (i.e., disease scores ± SD) or assayed⁷ for total levels of Verticillium DNA (i.e., ng/g plant tissue ± SD) in the stems (lower). In mixed infections levels for each fungus also were determined (black and white bars, respectively). Results summarize the data for 9–12 plants per time point for each interaction.These results demonstrate that Dvd-E6 infection protects Craigella tomatoes against colonization by and symptom development from Vd1 but not Vaa. Past studies often have suggested that the protective effect stems from an endophyte-induced activation of systemic acquired resistance (SAR) in the host providing protection against a broad range of pathogens.¹⁰ However, in the tolerant CS//Dvd-E6 interaction the protective effect appears to be targeted more directly, allowing Dvd-E6 to effectively restrict it''s virulent cousin, Vd1. In this context, it may be important that both of the Verticillium dahliae isolates from tomato are endemic to Ontario^7,11 and potentially in direct competition, while Verticillium albo-atrum from tomato is not. Previous molecular analyses suggested that the protective effect provided by Dvd-E6 colonization requires a genetic interplay with the host, selectively modifying the expression of specific tomato genes to be detrimental to Vd1. The experimental results presented here provide evidence that Verticillium-induced protection is restricted in range.     

Improved salt tolerance in tobacco plants by co-transformation of a betaine synthesis gene <Emphasis Type="Italic">BADH</Emphasis> and a vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene <Emphasis Type="Italic">SeNHX1</Emphasis>

Three types of transgenic tobacco plants were acquired by separate transformation or co-transformation of a vacuolar Na⁺/H⁺ antiporter gene, SeNHX1, and a betaine synthesis gene, BADH. When exposed to 200 mM NaCl, the dual gene-transformed plants displayed greater accumulation of betaine and Na⁺ than their wild-type counterparts. Photosynthetic rate and photosystem II activity in the transgenic plants were less affected by salt stress than wild-type plants. Transgenic plants exhibited a greater increase in osmotic pressure than wild-type plants when exposed to NaCl. More importantly, the dual gene transformed plants accumulated higher biomass than either of the single transgenic plants under salt stress. Taken together, these findings indicate that simultaneous transformation of BADH and SeNHX1 genes into tobacco plants can enable plants to accumulate betaine and Na⁺, thus conferring them more tolerance to salinity than either of the single gene transformed plants or wild-type tobacco plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The <Emphasis Type="Italic">ABI2</Emphasis>-dependent abscisic acid signalling controls HrpN-induced drought tolerance in <Emphasis Type="Italic">Arabidopsis</Emphasis>

HrpN, a protein produced by the plant pathogenic bacterium Erwinia amylovora, has been shown to stimulate plant growth and resistance to pathogens and insects. Here we report that HrpN activates abscisic acid (ABA) signalling to induce drought tolerance (DT) in Arabidopsis thaliana L. plants grown with water stress. Spraying wild-type plants with HrpN-promoted stomatal closure decreased leaf transpiration rate, increased moisture and proline levels in leaves, and alleviated extents of damage to cell membranes and plant drought symptoms caused by water deficiency. In plants treated with HrpN, ABA levels increased; expression of several ABA-signalling regulatory genes and the important effector gene rd29B was induced or enhanced. Induced expression of rd29B, promotion of stomatal closure, and reduction in drought severity were observed in the abi1-1 mutant, which has a defect in the phosphatase ABI1, after HrpN was applied. In contrast, HrpN failed to induce these responses in the abi2-1 mutant, which is impaired in the phosphatase ABI2. Inhibiting wild-type plants to synthesize ABA eliminated the role of HrpN in promoting stomatal closure and reducing drought severity. Moreover, resistance to Pseudomonas syringae developed in abi2-1 as in wild-type plants following treatment with HrpN. Thus, an ABI2-dependent ABA signalling pathway is responsible for the induction of DT but does not affect pathogen defence under the circumstances of this study.Hong-Ping Dong and Haiqin Yu contributed equally to this study and are regarded as joint first authors.     

Callus,shoot and hairy root formation in vitro as affected by the sensitivity to auxin and ethylene in tomato mutants

We analyzed the impact of ethylene and auxin disturbances on callus, shoots and Agrobacterium rhizogenes-induced hairy root formation in tomato (Solanum lycopersicum L.). The auxin low-sensitivity dgt mutation showed little hairy root initiation, whereas the ethylene low-sensitivity Nr mutation did not differ from the control Micro-Tom cultivar. Micro-Tom and dgt hairy roots containing auxin sensitivity/biosynthesis rol and aux genes formed prominent callus onto media supplemented with cytokinin. Under the same conditions, Nr hairy roots did not form callus. Double mutants combining Rg1, a mutation conferring elevated shoot formation capacity, with either dgt or Nr produced explants that formed shoots with little callus proliferation. The presence of rol + aux genes in Rg1 hairy roots prevented shoot formation. Taken together, the results suggest that although ethylene does not affect hairy root induction, as auxin does, it may be necessary for auxin-induced callus formation in tomato. Moreover, excess auxin prevents shoot formation in Rg1.     

Programmed cell death suppression in transformed plant tissue by tomato cDNAs identified from an <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-based functional screen

The genetic regulation of programmed cell death (PCD) is well characterized in animal systems, but largely unresolved in plants. This research was designed to identify plant genes that can suppress PCD triggered in plants by Fumonisin B1 (FB1). Agrobacterium rhizogenes was used to transform individual members of a cDNA library into tomato roots, which were then screened for resistance to FB1. Cellular changes elicited during FB1-induced PCD include chromatin condensation, fragmentation into pycnotic DNA bodies, TUNEL positive reactions, ROS accumulation, and eventual loss of membrane integrity. Several cDNA library members collectively overexpressed in a transformed root population revealed PCD suppressive action and were recovered by PCR. One of the FB1 suppressive genes was homologous to metallothionein, and shared sequence homology to the animal ortholog reported to suppress PCD through interference with formation or activity of reactive oxygen species (ROS). The metallothionein recovered in this screen suppressed ROS accumulation in FB1-treated roots and prevented symptoms of PCD. Anti-PCD genes recovered by this screen represent potential sources of resistance to PCD-dependent plant diseases, while the screen should be useful to identify genes capable of suppressing PCD triggered by other effectors, including those expressed by root pathogens during infection.     

Gene suppression in a tolerant tomato–vascular pathogen interaction

A plant can respond to the threat of a pathogen through resistance defenses or through tolerance. Resistance has been widely studied in many host pathogen systems but little is known about genetic changes which underlie a tolerant interaction. A recently developed model system for a tolerant tomato (Lycopersicon esculentum Mill) interaction with a fungal wilt pathogen, Verticillium dahliae Kleb, is examined with respect to changes in gene expression and compared to a susceptible infection. The results indicate that genetic changes can be dramatically different and some genes that are strongly elevated in the susceptible interaction are actually down-regulated in tolerance. Similar levels of fungal DNA and an up-regulation of many pathogenesis related genes indicate that in both types of interaction the presence of fungus is clearly recognized by the plant but other changes correlate with the absence of symptoms in the tolerant interaction. For example, a gene encoding a known 14-3-3 regulatory protein and a number of genes normally affected by this protein are down-regulated. Furthermore, genes which may contribute to foliar necrosis and cell death in the susceptible interaction also appear to be suppressed in the tolerant interaction, raising the possibility that the wilt symptoms, chlorosis and necrosis which are observed in the susceptible interaction, are actually programmed to further limit the growth of the fungal pathogen, and protect the general tomato population.     

Mapping a new nematode resistance locus in Lycopersicon peruvianum

Accessions of the wild tomato species L. peruvianum were screened with a root-knot nematode population (557R) which infects tomato plants carrying the nematode resistance gene Mi. Several accessions were found to carry resistance to 557R. A L. peruvianum backcross population segregating for resistance to 557R was produced. The segregation ratio of resistant to susceptible plants suggested that a single, dominant gene was a major factor in the new resistance. This gene, which we have designated Mi-3, confers resistance against nematode strains that can infect plants carrying Mi. Mi-3, or a closely linked gene, also confers resistance to nematodes at 32°C, a temperature at which Mi is not effective. Bulked-segregant analysis with resistant and susceptible DNA pools was employed to identify RAPD markers linked to this gene. Five-hundred-and-twenty oligonucleotide primers were screened and two markers linked to the new resistance gene were identified. One of the linked markers (NR14) was mapped to chromosome 12 of tomato in an L. esculentum/L. pennellii mapping population. Linkage of NR14 and Mi-3 with RFLP markers known to map on the short arm of chromosome 12 was confirmed by Southern analysis in the population segregating for Mi-3. We have positioned Mi-3 near RFLP marker TG180 which maps to the telomeric region of the short arm of chromosome 12 in tomato.     

Effects of Foliar and Root Applications of Methanol on the Growth of Arabidopsis, Tobacco, and Tomato Plants

The effects of aqueous methanol solutions applied as a foliar spray or via irrigation were investigated in Arabidopsis, tobacco, and tomato plants. Methanol applied to roots leads to phytotoxic damage in all three species tested. Foliar application causes an increase of fresh and dry weight in Arabidopsis and tobacco plants, but not in tomato plants. The increase in fresh and dry weight of Arabidopsis plants does not correlate with increased levels of soluble sugars, suggesting that increased accumulation of other products is responsible for the differences in the methanol-treated leaves. Foliar application of methanol can induce pectin methylesterase (PME) gene expression in Arabidopsis and tomato plants, activating specific PME genes.     

The diageotropica mutation alters auxin induction of a subset of the Aux/IAA gene family in tomato

The diageotropica (dgt) mutation has been proposed to affect either auxin perception or responsiveness in tomato plants. It has previously been demonstrated that the expression of one member of the Aux/IAA family of auxin-regulated genes is reduced in dgt plants. Here, we report the cloning of ten new members of the tomato Aux/IAA family by PCR amplification based on conserved protein domains. All of the gene family members except one (LeIAA7) are expressed in etiolated tomato seedlings, although they demonstrate tissue specificity (e.g. increased expression in hypocotyls vs. roots) within the seedling. The wild-type auxin-response characteristics of the expression of these tomato LeIAA genes are similar to those previously described for Aux/IAA family members in Arabidopsis. In dgt seedlings, auxin stimulation of gene expression was reduced in only a subset of LeIAA genes (LeIAA5, 8, 10, and 11), with the greatest reduction associated with those genes with the strongest wild-type response to auxin. The remaining LeIAA genes tested exhibited essentially the same induction levels in response to the hormone in both dgt and wild-type hypocotyls. These results confirm that dgt plants can perceive auxin and suggest that a specific step in early auxin signal transduction is disrupted by the dgt mutation.     

Salicylic-Acid-Induced Self-excision of the Marker Gene <Emphasis Type="Italic">npt</Emphasis>II from Transgenic Tomato Using the Cre–<Emphasis Type="Italic">loxP</Emphasis> System

The presence of antibiotic-resistant genes in genetically engineered crops together with the target gene has generated a number of environmental and consumer concerns. In order to alleviate public concerns over the safety of food derived from transgenic crops, marker gene elimination is desirable. Marker-free transgenic tomato plants were obtained by using a salicylic-acid-regulated Cre–loxP-mediated site-specific DNA recombination system in which the selectable marker neomycin phosphotransferase nptII and cre genes were flanked by two directly oriented loxP sites. Upon induction by salicylic acid, the cre gene produced a recombinase that eliminated sequences encoding nptII and cre genes, sandwiched by two loxP sites from the tomato genome. Regenerant plants with the Cre–loxP system were obtained by selection on kanamycin media and polymerase chain reaction (PCR) screening. Transgenic plants were screened for excision by PCR using nptII, cre, and PR-1a promoter primers following treatment with salicylic acid. The footprint of the excision was determined by sequencing the T-DNA borders after a perfect recombination event. The excision efficiency was 38.7%. A new plant transformation vector, pBLNSC (Genbank accession number EU327497), was developed, containing six cloning sites and the self-excision system. This provided an effective approach to eliminate the selectable marker gene from transgenic tomato, thus expediting public acceptance of genetically modified tomato.     

Constitutive expression of a meiotic recombination protein gene homolog, OsTOP6A1, from rice confers abiotic stress tolerance in transgenic Arabidopsis plants

Plant productivity is greatly influenced by various environmental stresses, such as high salinity and drought. Earlier, we reported the isolation of topoisomerase 6 homologs from rice and showed that over expression of OsTOP6A3 and OsTOP6B confers abiotic stress tolerance in transgenic Arabidopsis plants. In this study, we have assessed the function of nuclear-localized topoisomerase 6 subunit A homolog, OsTOP6A1, in transgenic Arabidopsis plants. The over expression of OsTOP6A1 in transgenic Arabidopsis plants driven by cauliflower mosaic virus-35S promoter resulted in pleiotropic effects on plant growth and development. The transgenic Arabidopsis plants showed reduced sensitivity to stress hormone, abscisic acid (ABA), and tolerance to high salinity and dehydration at the seed germination; seedling and adult stages as reflected by the percentage of germination, fresh weight of seedlings and leaf senescence assay, respectively. Concomitantly, the expression of many stress-responsive genes was enhanced under various stress conditions in transgenic Arabidopsis plants. Moreover, microarray analysis revealed that the expression of a large number of genes involved in various processes of plant growth and development and stress responses was altered in transgenic plants. Although AtSPO11-1, the homolog of OsTOP6A1 in Arabidopsis, has been implicated in meiotic recombination; the present study demonstrates possible additional role of OsTOP6A1 and provides an effective tool for engineering crop plants for tolerance to different environmental stresses. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Immunocytochemical localization of cytokinins in Craigella tomato and a sideshootless mutant

Post-embedding immunocytochemical techniques using peroxidase-antiperoxidase or immunoglobulin G-gold as markers were used for the localization of cytokinins (CKs) in two isogenic lines, Craigella (C) and Craigella lateral suppressor (Cls), of tomato Lycopersicon esculentum Mill. Terminal buds, nodes, hypocotyl segments and root tips were submitted to a periodate-borohydride procedure, to obtain the coupling of isopentenyladeosine and zeatin riboside to cellular proteins, followed by a fixative step with a paraformaldehyde and glutaraldehyde mixture. Enzyme-linked immunosorbent assay tests performed on ovalbumin-coated microtitration plates have shown that this method was effective for CK riboside and base coupling to proteins. Paraffin-wax- or Spurr's-resin-embedded sections were cleared of wax or resin before incubation with anti-zeatin riboside or anti-isopentenyladenosine antibodies. The procedure was thoroughly investigated and many controls were done in order to eliminate artefacts. The immunostaining patterns observed along the plants showed a basipetally decreasing gradient of CKs along the stem and in the roots. Immunolabelling was higher in the actively growing regions of the stem bud and root apices. Terminal buds of Cls appeared to be less immunoreactive than C, whereas no differences were detected in root-tip immunolabelling. The staining patterns are consistent with the idea that root and bud apices have a different CK metabolism. The absence of axillary bud formation in Cls is correlated with low CK levels in the organogensis sites.Abbreviations C Craigella, isogenic line - CK cytokinin - Cls Craigella lateral suppressor - EDC 1-(3-dimethylaminopropyl)3-ethylcarbodiimide hydrochloride - ELISA enzyme-linked immunosorbent assay - 2iP isopentenyladenine - 2iPA isopentenyladenosine - PAP peroxidase-anti-peroxidase - PFAG paraformaldehyde/glutaraldehyde mixture - Z zeatin - ZR zeatin riboside     

Negative regulation of <Emphasis Type="Italic">KNOX</Emphasis> expression in tomato leaves

Leaves of seed plants can be described as simple, where the leaf blade is entire, or dissected, where the blade is divided into distinct leaflets. Both simple and dissected leaves are initiated at the flanks of a pluripotent structure termed the shoot apical meristem (SAM). In simple-leafed species, expression of class I KNOTTED1-like homeobox (KNOX) proteins is confined to the meristem while in many dissected leaf plants, including tomato, KNOX expression persists in leaf primordia. Elevation of KNOX expression in tomato leaves can result in increased leaflet number, indicating that tight regulation of KNOX expression may help define the degree of leaf dissection in this species. To test this hypothesis and understand the mechanisms controlling leaf dissection in tomato, we studied the clausa (clau) and tripinnate (tp) mutants both of which condition increased leaflet number phenotypes. We show that TRIPINNATE and CLAUSA act together, to restrict the expression level and domain of the KNOX genes Tkn1 and LeT6/Tkn2 during tomato leaf development. Because loss of CLAU or TP activity results in increased KNOX expression predominantly on the adaxial (upper) leaf domain, our observations indicate that CLAU and TP may participate in a domain-specific KNOX repressive system that delimits the ability of the tomato leaf to generate leaflets.     

Interaction of Glomus mosseae and Paecilomyces lilacinus on Meloidogyne javanica of tomato

The effects of Glomus mosseae and Paecilomyces lilacinus on Meloidogyne javanica of tomato were tested in a greenhouse experiment. Chicken layer manure was used as a carrier substrate for the inoculum of P. lilacinus. The following parameters were used: gall index, average number of galls per root system, plant height, shoot and root weights. Inoculation of tomato plants with G. mosseae did not markedly increase the growth of infected plants with M. javanica. Inoculation of plants with G. mosseae and P. lilacinus together or separately resulted in similar shoots and plant heights. The highest root development was achieved when mycorrhizal plants were inoculated with P. lilacinus to control root-knot nematode. Inoculation of tomato plants with G. mosseae suppressed gall index and the average number of galls per root system by 52% and 66%, respectively, compared with seedlings inoculated with M. javanica alone. Biological control with both G. mosseae and P. lilacinus together or separately in the presence of layer manure completely inhibited root infection with M. javanica. Mycorrhizal colonization was not affected by the layer manure treatment or by root inoculation with P. lilacinus. Addition of layer manure had a beneficial effect on plant growth and reduced M. javanica infection.     

Genetic transformation of sweet orange with the coat protein gene of <Emphasis Type="Italic">Citrus psorosis virus</Emphasis> and evaluation of resistance against the virus

Citrus psorosis is a serious viral disease affecting citrus trees in many countries. Its causal agent is Citrus psorosis virus (CPsV), the type member of genus Ophiovirus. CPsV infects most important citrus varieties, including oranges, mandarins and grapefruits, as well as hybrids and citrus relatives used as rootstocks. Certification programs have not been sufficient to control the disease and no sources of natural resistance have been found. Pathogen-derived resistance (PDR) can provide an efficient alternative to control viral diseases in their hosts. For this purpose, we have produced 21 independent lines of sweet orange expressing the coat protein gene of CPsV and five of them were challenged with the homologous CPV 4 isolate. Two different viral loads were evaluated to challenge the transgenic plants, but so far, no resistance or tolerance has been found in any line after 1 year of observations. In contrast, after inoculation all lines showed characteristic symptoms of psorosis in the greenhouse. The transgenic lines expressed low and variable amounts of the cp gene and no correlation was found between copy number and transgene expression. One line contained three copies of the cp gene, expressed low amounts of the mRNA and no coat protein. The ORF was cytosine methylated suggesting a PTGS mechanism, although the transformant failed to protect against the viral load used. Possible causes for the failed protection against the CPsV are discussed.