Identification of a Protein that Interacts with LeATL6 Ubiquitin‐protein Ligase E3 Upregulated in Tomato Treated with Elicitin‐Like Cell Wall Proteins of Pythium oligandrum

The expression of LeATL6, which encodes RING‐H2 zinc finger ubiquitin‐protein ligase E3, is highly induced in tomato roots treated with the elicitin‐like cell wall protein fraction (CWP) from the non‐pathogenic oomycete Pythium oligandrum, which enhances resistance to pathogens through a jasmonic acid (JA)‐dependent signalling pathway. In this study, the role of LeATL6 for CWP‐induced defence response was further analysed. To screen the putative target protein of LeATL6 for the CWP‐induced defence mechanism in tomato, we used a yeast two‐hybrid system to screen five clones encoding a protein that interacts with LeATL6. Four clones had a function associated with the ubiquitin‐proteasome system. Another positive clone encoded a protein sharing homology with S‐adenosylmethionine decarboxylase (SAMDC). In CWP‐treated tomato roots, SAMDC activity was clearly suppressed. Thus, the interaction of SAMDC with LeATL6 and the decreased SAMDC activity may be associated with JA‐dependent induced resistance in tomato treated with P. oligandrum.     

Properties of capsaicinoids for the control of fungi and oomycetes pathogenic to pepper

Capsaicinoids are pungent compounds found in pepper (Capsicum spp.) fruits. Capsaicin showed antimicrobial activity in plate assays against seven isolates of five species of fungi and nine isolates of two species of oomycetes. The general trend was that oomycetes were more inhibited than fungi. Assays of capsaicin biosynthetic precursors suggest that the lateral chain of capsaicinoids has more inhibitory activity than the phenolic part. In planta tests of capsaicinoids (capsaicin and N‐vanillylnonanamide) applied to the roots demonstrated that these compounds conferred protection against the pathogenic fungus Verticillium dahliae and induced both chitinase activity and expression of several defence‐related genes, such as CASC1, CACHI2 and CABGLU. N‐Vanillylnonanamide infiltrated into cotyledons confers systemic protection to the upper leaves of pepper against the fungal pathogen Botrytis cinerea. In wild‐type tomato plants such cotyledon infiltration has no protective effect, but is effective in the Never‐ripe tomato mutant impaired in ethylene response. A similar effect was observed in tomato after salicylic acid infiltration.     

Feeding‐induced changes in defence enzymes and PR proteins and their implications in host resistance to Nilaparvata lugens

Six rice genotypes showing susceptible and resistant reactions to brown planthopper (BPH), Nilaparvata lugens were studied for feeding‐induced changes in defence enzymes and pathogenesis‐related (PR) proteins. The high resistant genotypes PTB 33, ADT 45 and ASD 7 and moderately resistant genotypes CO 43 and KAU 1661 recorded the greater expression of defence enzymes peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, total phenol and β‐1,3 glucanase in response to N. lugens feeding at 1 day after infestation (DAI) compared with susceptible genotype TN1. The greater activity of chitinase was observed in resistant cultivars at 3 DAI and the activity was sustained for more than 1 week compared with susceptible TN1. In conclusion, the current study revealed that these defence enzymes and PR proteins might attribute to the resistance mechanisms in rice plants against BPH infestation.     

Ample genetic variation but no evidence for genotype specificity in an all‐parthenogenetic host–parasitoid interaction

Antagonistic coevolution between hosts and parasites can result in negative frequency‐dependent selection and may thus be an important mechanism maintaining genetic variation in populations. Negative frequency‐dependence emerges readily if interactions between hosts and parasites are genotype‐specific such that no host genotype is most resistant to all parasite genotypes, and no parasite genotype is most infective on all hosts. Although there is increasing evidence for genotype specificity in interactions between hosts and pathogens or microparasites, the picture is less clear for insect host–parasitoid interactions. Here, we addressed this question in the black bean aphid (Aphis fabae) and its most important parasitoid Lysiphlebus fabarum. Because both antagonists are capable of parthenogenetic reproduction, this system allows for powerful tests of genotype × genotype interactions. Our test consisted of exposing multiple host clones to different parthenogenetic lines of parasitoids in all combinations, and this experiment was repeated with animals from four different sites. All aphids were free of endosymbiotic bacteria known to increase resistance to parasitoids. We observed ample genetic variation for host resistance and parasitoid infectivity, but there was no significant host clone × parasitoid line interaction, and this result was consistent across the four sites. Thus, there is no evidence for genotype specificity in the interaction between A. fabae and L. fabarum, suggesting that the observed variation is based on rather general mechanisms of defence and attack.     

Design of a bacterial speck resistant tomato by CRISPR/Cas9‐mediated editing of SlJAZ2

Due to their different lifestyles, effective defence against biotrophic pathogens normally leads to increased susceptibility to necrotrophs, and vice versa. Solving this trade‐off is a major challenge for obtaining broad‐spectrum resistance in crops and requires uncoupling the antagonism between the jasmonate (JA) and salicylate (SA) defence pathways. Pseudomonas syringae pv. tomato (Pto) DC3000, the causal agent of tomato bacterial speck disease, produces coronatine (COR) that stimulates stomata opening and facilitates bacterial leaf colonization. In Arabidopsis, stomata response to COR requires the COR co‐receptor AtJAZ2, and dominant AtJAZ2Δjas repressors resistant to proteasomal degradation prevent stomatal opening by COR. Here, we report the generation of a tomato variety resistant to the bacterial speck disease caused by PtoDC3000 without compromising resistance to necrotrophs. We identified the functional ortholog of AtJAZ2 in tomato, found that preferentially accumulates in stomata and proved that SlJAZ2 is a major co‐receptor of COR in stomatal guard cells. SlJAZ2 was edited using CRISPR/Cas9 to generate dominant JAZ2 repressors lacking the C‐terminal Jas domain (SlJAZ2Δjas). SlJAZ2Δjas prevented stomatal reopening by COR and provided resistance to PtoDC3000. Water transpiration rate and resistance to the necrotrophic fungal pathogen Botrytis cinerea, causal agent of the tomato gray mold, remained unaltered in Sljaz2Δjas plants. Our results solve the defence trade‐off in a crop, by spatially uncoupling the SA‐JA hormonal antagonism at the stomata, entry gates of specific microbes such as PtoDC3000. Moreover, our results also constitute a novel CRISPR/Cas‐based strategy for crop protection that could be readily implemented in the field.     

Role of gibberellic acid in tomato defence against potato purple top phytoplasma infection

Infection of tomato by potato purple top (PPT) phytoplasma causes disruption of gibberellin (GA) homeostasis in the plant host. Such pathologically‐induced GA deficiency can be partially reversed by exogenous application of GA. This study was designed to explore the role of GA in tomato defence response against phytoplasmal disease, and to determine whether pretreatment with GA would protect healthy tomato seedlings from subsequent phytoplasmal infection and disease development. Our results revealed that, following exogenous GA application and subsequent PPT phytoplasma graft inoculation, there was an apparently coordinated down‐regulation of the gene encoding a key GA signalling component and growth repressor known as DELLA protein (GAI) and up‐regulation of genes involved in salicylic acid (SA) synthesis (ICS1), signalling (NIM1) and downstream defence responses (PRP‐1). Our results also indicated that differential regulation of the above genes was correlated with an increase in activities of defence‐related enzymes β‐1,3‐glucanase and chitinase. The data presented in this communication provide evidence to suggest that GA may act via DELLA and SA signalling pathways to modulate host defence in response to PPT phytoplasma infection. Although the GA pretreatment‐induced defence was not sufficient to prevent a systemic infection, it reduced phytoplasma titre and significantly attenuated disease symptoms. While the actual molecular mechanism underlying the GA‐induced plant defence remains elusive, findings from the current study open new opportunities for in‐depth studies of the functional role of the GA signalling network during defence response against phytoplasmal infection.     

Sl‐lncRNA15492 interacts with Sl‐miR482a and affects Solanum lycopersicum immunity against Phytophthora infestans

Long non‐coding RNAs (lncRNAs) are involved in the resistance of plants to infection by pathogens via interactions with microRNAs (miRNAs). Long non‐coding RNAs are cleaved by miRNAs to produce phased small interfering RNAs (phasiRNAs), which, as competing endogenous RNAs (ceRNAs), function as decoys for mature miRNAs, thus inhibiting their expression, and contain pre‐miRNA sequences to produce mature miRNAs. However, whether lncRNAs and miRNAs mediate other molecular mechanisms during plant resistance to pathogens is unknown. In this study, as a positive regulator, Sl‐lncRNA15492 from tomato (Solanum lycopersicum Zaofen No. 2) plants affected tomato resistance to Phytophthora infestans. Gain‐ and loss‐of‐function experiments and RNA ligase‐mediated 5′‐amplification of cDNA ends (RLM‐5′ RACE) also revealed that Sl‐miR482a was negatively involved in tomato resistance by targeting Sl‐NBS‐LRR genes and that silencing of Sl‐NBS‐LRR1 decreased tomato resistance. Sl‐lncRNA15492 inhibited the expression of mature Sl‐miR482a, whose precursor was located within the antisense sequence of Sl‐lncRNA15492. Further degradome analysis and additional RLM‐5′ RACE experiments verified that mature Sl‐miR482a could also cleave Sl‐lncRNA15492. These results provide a mechanism by which lncRNAs might inhibit precursor miRNA expression through antisense strands of lncRNAs, and demonstrate that Sl‐lncRNA15492 and Sl‐miR482a mutually inhibit the maintenance of Sl‐NBS‐LRR1 homeostasis during tomato resistance to P. infestans.     

Transgenic tomato plants expressing a wheat endochitinase gene demonstrate enhanced resistance to <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis>

Various chitinases have been shown to inhibit the growth of fungal pathogens in in vitro as well as in planta conditions. chi194, a wheat chitinases gene encoding a 33-kDa chitinase protein, was overexpressed in tomato plants (cv. Pusa Ruby) under the control of maize ubiquitin 1 promoter. The integration of transgene in tomato plants was confirmed with polymerase chain reaction (PCR) and Southern blot analysis. The inheritance of the transgene in T₁ and T₂ generations were shown by molecular analysis and the hygromycin sensitivity test. The broad range of chitinase activity was observed among the transgenic lines in T₀ and a similar range was retained in the T₁ and T₂ generations. Most importantly, the transgenic tomato lines with high chitinase activity were found to be highly resistant to the fungal pathogen Fusarium oxysporum f. sp. lycopersici. Thus, the results demonstrated that the expression of the wheat endochitinase chi194 in tomato plants confers resistance against Fusarium wilt disease caused by the fungal pathogen Fusarium oxysporum f. sp. lycopersici.     

Comparative analysis reveals changes in transcriptomes of sugarcane upon infection by Leifsoniaxyli subsp. xyli

Ratoon stunting disease (RSD) caused by bacterium Leifsoniaxyli subsp. xyli (Lxx) is a devastating disease of sugarcane over a large part of the world. Genetic improvement for RSD‐resistant varieties is considered the most effective method to control the disease. However, genetic improvement of sugarcane is hindered by the limited information about the molecular mechanisms underlying Lxx pathogenicity and defence responses in sugarcane. In this study, genome‐wide gene expression profiling was used to compare RSD‐resistant (CP72‐2086) and RSD‐susceptible (GT11) genotypes at different infection time points in order to identify the candidate regulators for RSD resistance. A total of 14,494 differentially expressed genes (DEGs) were identified, indicating that dramatic changes had occurred in gene expression upon Lxx infection, especially in the susceptible genotype. Enrichment analysis showed that a large number of genes related to plant hormone signal transduction, phenylalanine metabolism, phenylpropanoid biosynthesis and starch and sucrose metabolism was responsible for sugarcane response to Lxx infection. Plant hormone signalling pathway genes were significantly differentially expressed at the early infection stage between the two genotypes. The resistant genotype chose the jasmonic acid‐ and ethylene‐dependent host‐defence pathways to resist Lxx infection, whereas the susceptible genotype preferred the salicylic acid‐dependent host‐defence pathways. These findings help unravel the molecular mechanisms of sugarcane plant–Lxx interactions and may pave the way for sugarcane breeding for disease resistance.     

Floral nectar of the obligate outcrossing Canavalia gladiata (Jacq.) DC. (Fabaceae) contains only one predominant protein,a class III acidic chitinase

• Floral nectar can affect the fitness of insect‐pollinated plants, through both attraction and manipulation of pollinators. Self‐incompatible insect‐pollinated plants receive more insect visits than their self‐compatible relatives, and the nectar of such species might face increased risk of infestation by pathogens carried by pollinators than self‐compatible plants. Proteins in nectar (nectarins) play an important role in protecting the nectar, but little is known regarding nectarins in self‐incompatible species.
• The nectarins from a self‐incompatible and insect‐pollinated leguminous crop, Canavalia gladiata, were separated using two‐dimensional electrophoresis and analysed using mass spectrometry. The predominant nectarin gene was cloned and the gene expression pattern investigated using quantitative real‐time PCR. Chitinolytic activity in the nectar was tested with different substrates.
• The C. gladiata nectar proteome only has one predominant nectarin, an acidic class III chitinase (CaChi3). The full‐length CaChi3 gene was cloned, coding for a protein of 298 amino acids with a predicted signal peptide. CaChi3 is very similar to members of the class III chitinase family, whose evolution is dominated by purifying selection. CaChi3 was expressed in both nectary and leaves. CaChi3 has thermostable chitinolytic activity according to glycol‐chitin zymography or a fluorogenic substratem but has no lysozyme activity.
• Chitinase might be a critical protein component in nectar. The extremely simple nectar proteome in C. gladiata disproves the hypothesis that self‐incompatible species always have more complex nectar proteomes. Accessibility of nectar might be a significant determinant of the evolutionary pressure to develop nectar defence mechanisms.

     

Potential of bioinsecticidal Bacillus thuringiensis inoculum to suppress gray mold in tomato based on induced systemic resistance

The potential of the active ingredient of a commercial bioinsecticide, XenTari^® (Bacillus thuringiensis [BT] serovar aizawai strain ABTS‐1857), to suppress gray mold in tomato plants was elucidated. First, a suspension of the active ingredient of XenTari^® and a liquid culture of the bacterial strain as BT inocula were sprayed onto detached leaves or drenched into pots of tomato seedlings, and then, propagules of the gray mold fungus, Botrytis cinerea, were inoculated onto the leaves. The gray mold disease was significantly suppressed when rhizospheres were drenched with either inoculum, but not when inocula were sprayed onto detached leaves of seedlings. Both BT inocula were verified not to directly inhibit the mycelial growth of B. cinerea based on in vitro culture plate assays. Additionally, real‐time RT‐PCR analysis verified that the active ingredient increased the expression levels of defence‐related genes, such as PR‐1(P6) and P4, in the leaves of tomato seedlings. These results suggest that the active ingredient has the potential to suppress gray mold disease in tomato, not through direct antagonistic interactions with B. cinerea, but rather through systemic activation of the plant defence system by increased expression of several defence‐related genes.     

Ectopic Expression of Rice OsBIANK1, Encoding an Ankyrin Repeat‐Containing Protein,in Arabidopsis Confers Enhanced Disease Resistance to Botrytis cinerea and Pseudomonas syringae

Ankyrin repeat‐containing proteins comprise a large family whose members have been shown to play important roles in various aspects of biological processes in plant growth and development as well as in responses to biotic and abiotic stresses. We previously identified a rice gene, OsBIANK1, encoding an ankyrin repeat‐containing protein and found that expression of OsBIANK1 can be induced by defence signalling molecules and by infection of Magnaporthe oryzae, the causal agent of blast disease. To better understand the possible function of OsBIANK1 in disease resistance, we generated transgenic Arabidopsis plants that constitutively overexpress the OsBIANK1 gene. Results from disease assays revealed that the OsBIANK1‐overexpressing plants display increased resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000 as compared with the wild‐type plants. In OsBIANK1‐overexpressing plants, expression of some of well‐known defence genes (e.g. PR‐1, PR‐2 and PDF1.2) was up‐regulated after infection with B. cinerea or P. syringae pv. tomato DC3000. Furthermore, the OsBIANK1‐overexpressing plants showed decreased levels of reactive oxygen species (i.e. superoxide anion and H₂O₂) after Botrytis infection. Thus, our present results further support the role of OsBIANK1 in regulation of defence responses against different types of pathogens.     

An untargeted global metabolomic analysis reveals the biochemical changes underlying basal resistance and priming in Solanum lycopersicum,and identifies 1‐methyltryptophan as a metabolite involved in plant responses to Botrytis cinerea and Pseudomonas syringae

In this study, we have used untargeted global metabolomic analysis to determine and compare the chemical nature of the metabolites altered during the infection of tomato plants (cv. Ailsa Craig) with Botrytis cinerea (Bot) or Pseudomonas syringae pv. tomato DC3000 (Pst), pathogens that have different invasion mechanisms and lifestyles. We also obtained the metabolome of tomato plants primed using the natural resistance inducer hexanoic acid and then infected with these pathogens. By contrasting the metabolomic profiles of infected, primed, and primed + infected plants, we determined not only the processes or components related directly to plant defense responses, but also inferred the metabolic mechanisms by which pathogen resistance is primed. The data show that basal resistance and hexanoic acid‐induced resistance to Bot and Pst are associated with a marked metabolic reprogramming. This includes significant changes in amino acids, sugars and free fatty acids, and in primary and secondary metabolism. Comparison of the metabolic profiles of the infections indicated clear differences, reflecting the fact that the plant's chemical responses are highly adapted to specific attackers. The data also indicate involvement of signaling molecules, including pipecolic and azelaic acids, in response to Pst and, interestingly, to Bot. The compound 1‐methyltryptophan was shown to be associated with the tomato–Pst and tomato–Bot interactions as well as with hexanoic acid‐induced resistance. Root application of this Trp‐derived metabolite also demonstrated its ability to protect tomato plants against both pathogens.     

Tuber Blight Development in Potato Cultivars in Response to Different Genotypes of Phytophthora infestans

Migrations or introduction of new genotypes of Phytophthora infestans to a specific region imposes a different perspective for potato production. During 2009–2010, a late blight epidemic affected the Northeastern United States, which quickly spread through several states. The epidemic was characterized by the appearance of a new genotype of P. infestans designated US‐22, which was isolated from tomato and potato. Potato tubers are an essential component of late blight epidemics where the pathogen cannot overwinter on Solanaceous plants. Six potato cultivars were inoculated with 12 isolates of P. infestans (five different genotypes), including isolates of the genotype US‐22. Tuber blight development was characterized in terms of tissue darkening expressed as area under the disease progress curve values and lenticel infection. The responses indicated that US‐8 was more aggressive than US‐22, but US‐22 isolates obtained from potato were more aggressive on potato than those acquired from tomato. Tuber periderm responses to infection were limited, yet US‐8 isolates infected the periderm more often than US‐22 isolates. There were significant differences among the cultivars tested but cv. Jacqueline Lee was the most resistant overall. Although isolates of P. infestans genotype US‐22 were less aggressive in comparison with US‐8 isolates, US‐22 isolates still infected potato tubers and were as aggressive us US‐8 isolates on some cultivars. Management of late blight caused by isolates of US‐22 through host resistance may be feasible but imposes a different set of criteria for consideration from those that US‐8 imposed.     

A novel receptor‐like kinase involved in fungal pathogen defence in Arabidopsis thaliana

Plants are under constant attack from a variety of disease‐causing organisms. Lacking an adaptive immune system, plants repel pathogen attack via an array of pathogen recognition machinery. Receptor‐like kinases (RLKs) are involved in the recognition of pathogen‐associated molecular patterns (PAMPs) and activate resistance pathways against broad classes of pathogens. We have identified powdery mildew‐resistant kinase 1, an Arabidopsis gene encoding an RLK that is highly induced by chitin at early time points and localizes to the plasma membrane. Knockout mutants in pmrk1 are more susceptible to both Golovinomyces cichoracearum and Plectosphaerella cucumerina. Our data show that PMRK1 is essential in early stages of defence against fungi and provide evidence that PMRK1 may be unique to chitin‐induced signalling pathways. The results of this study indicate that PMRK1 is a critical component of plant innate immunity against fungal pathogens.     

Do strigolactones contribute to plant defence?

Strigolactones are multifunctional molecules involved in several processes outside and within the plant. As signalling molecules in the rhizosphere, they favour the establishment of arbuscular mycorrhizal symbiosis, but they also act as host detection cues for root parasitic plants. As phytohormones, they are involved in the regulation of plant architecture, adventitious rooting, secondary growth and reproductive development, and novel roles are emerging continuously. In the present study, the possible involvement of strigolactones in plant defence responses was investigated. For this purpose, the resistance/susceptibility of the strigolactone‐deficient tomato mutant Slccd8 against the foliar fungal pathogens Botrytis cinerea and Alternaria alternata was assessed. Slccd8 was more susceptible to both pathogens, pointing to a new role for strigolactones in plant defence. A reduction in the content of the defence‐related hormones jasmonic acid, salicylic acid and abscisic acid was detected by high‐performance liquid chromatography coupled to tandem mass spectrometry in the Slccd8 mutant, suggesting that hormone homeostasis is altered in the mutant. Moreover, the expression level of the jasmonate‐dependent gene PinII, involved in the resistance of tomato to B. cinerea, was lower than in the corresponding wild‐type. We propose here that strigolactones play a role in the regulation of plant defences through their interaction with other defence‐related hormones, especially with the jasmonic acid signalling pathway.     

Xanthomonas euvesicatoria type III effector XopQ interacts with tomato and pepper 14–3–3 isoforms to suppress effector‐triggered immunity

Effector‐triggered immunity (ETI) to host‐adapted pathogens is associated with rapid cell death at the infection site. The plant‐pathogenic bacterium Xanthomonas euvesicatoria (Xcv) interferes with plant cellular processes by injecting effector proteins into host cells through the type III secretion system. Here, we show that the Xcv effector XopQ suppresses cell death induced by components of the ETI‐associated MAP kinase cascade MAPKKKα MEK2/SIPK and by several R/avr gene pairs. Inactivation of xopQ by insertional mutagenesis revealed that this effector inhibits ETI‐associated cell death induced by avirulent Xcv in resistant pepper (Capsicum annuum), and enhances bacterial growth in resistant pepper and tomato (Solanum lycopersicum). Using protein–protein interaction studies in yeast (Saccharomyces cerevisiae) and in planta, we identified the tomato 14–3–3 isoform SlTFT4 and homologs from other plant species as XopQ interactors. A mutation in the putative 14–3–3 binding site of XopQ impaired interaction of the effector with CaTFT4 in yeast and its virulence function in planta. Consistent with a role in ETI, TFT4 mRNA abundance increased during the incompatible interaction of tomato and pepper with Xcv. Silencing of NbTFT4 in Nicotiana benthamiana significantly reduced cell death induced by MAPKKKα. In addition, silencing of CaTFT4 in pepper delayed the appearance of ETI‐associated cell death and enhanced growth of virulent and avirulent Xcv, demonstrating the requirement of TFT4 for plant immunity to Xcv. Our results suggest that the XopQ virulence function is to suppress ETI and immunity‐associated cell death by interacting with TFT4, which is an important component of ETI and a bona fide target of XopQ.     

Emergence of 13_A2 Blue Lineage of Phytophthora infestans was Responsible for Severe Outbreaks of Late Blight on Tomato in South‐West India

Prior to 2007, late blight was not reported as a serious threat to tomato cultivation in India although the disease has been known on potato since 1953. During the July–December cropping season of 2009 and 2010, severe late blight epidemics were observed in Karnataka state of India, causing crop losses up to 100%. Nineteen Phytophthora isolates, recovered from late blight affected tomato tissues from different localities in Karnataka state between 2009 and 2010, were identified as Phytophthora infestans based on morphology, a similarity search of ITS sequences at GenBank and species‐specific PCR using PINF/ITS5 primer pair. The isolates were further assessed for metalaxyl sensitivity, mating type, mitochondrial DNA (mtDNA) haplotype, DNA fingerprinting patterns based on simple sequence repeats (SSR) and RFLPs using the RG57 probe and aggressiveness on tomato. All isolates were metalaxyl resistant, A2 mating type, mtDNA haplotype Ia and had identical SSR and RG57 fingerprints and highly aggressive on tomato. The phenotypic and genotypic characters of isolates examined in this study were found to be similar to that of 13_A2 genotype of P. infestans population reported in Europe. Thus, appearance of new population similar to 13_A2 genotype was responsible for severe late blight epidemics on tomato in South‐West India.