Search

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins with pathogen sensor activities have evolved to initiate immune signaling by activating helper NLRs. However, the mechanisms underpinning helper NLR activation by sensor NLRs remain poorly understood. Although coiled coil (CC) type sensor NLRs such as the Potato virus X disease resistance protein Rx have been shown to activate the oligomerization of their downstream helpers NRC2, NRC3 and NRC4, the domains involved in sensor-helper signaling are not known. Here, we used Agrobacterium tumefaciens-mediated transient expression in Nicotiana benthamiana to show that the nucleotide-binding (NB) domain within the NB-ARC of Rx is necessary and sufficient for oligomerization and immune signaling of downstream helper NLRs. In addition, the NB domains of the disease resistance proteins Gpa2 (cyst nematode resistance), Rpi-amr1, Rpi-amr3 (oomycete resistance) and Sw-5b (virus resistance) are also sufficient to activate their respective downstream NRC helpers. Using transient expression in the lettuce (Lactuca sativa), we show that Rx (both as full length or as NB domain truncation) and its helper NRC2 form a minimal functional unit that can be transferred from solanaceous plants (lamiids) to Campanulid species. Our results challenge the prevailing paradigm that NLR proteins exclusively signal via their N-terminal domains and reveal a signaling activity for the NB domain of NRC-dependent sensor NLRs. We propose a model in which helper NLRs can perceive the status of the NB domain of their upstream sensors., (© 2024 The Authors. New Phytologist © 2024 New Phytologist Foundation.)

Mitigation strategies to prevent microbial contamination of crops are lacking. We tested the hypothesis that induction of plant systemic resistance by biological (induced systemic resistance [ISR]) and chemical (systemic acquired resistance [SAR]) elicitors reduces endophytic colonization of leaves by Salmonella enterica serovars Senftenberg and Typhimurium. S . Senftenberg had greater endophytic fitness than S. Typhimurium in basil and lettuce. The apoplastic population sizes of serovars Senftenberg and Typhimurium in basil and lettuce, respectively, were significantly reduced approximately 10- to 100-fold by root treatment with microbial inducers of systemic resistance compared to H 2 O treatment. Rhodotorula glutinis effected the lowest population increases of S. Typhimurium in lettuce and S . Senftenberg in basil leaves, respectively 120- and 60-fold lower than those seen with the H 2 O treatment over 10 days postinoculation. Trichoderma harzianum and Pichia guilliermondii did not have any significant effect on S . Senftenberg in the basil apoplast. The chemical elicitors acidobenzolar- S -methyl and dl-β-amino-butyric acid inhibited S. Typhimurium multiplication in the lettuce apoplast 10- and 2-fold, respectively, compared to H 2 O-treated plants. All ISR and SAR inducers applied to lettuce roots in this study increased leaf expression of the defense gene PR1 , as did Salmonella apoplastic colonization in H 2 O-treated lettuce plants. Remarkably, both acidobenzolar- S -methyl upregulation and R. glutinis upregulation of PR1 were repressed by the presence of Salmonella in the leaves. However, enhanced PR1 expression was sustained longer and at greater levels upon elicitor treatment than by Salmonella induction alone. These results serve as a proof of concept that priming of plant immunity may provide an intrinsic hurdle against the endophytic establishment of enteric pathogens in leafy vegetables. IMPORTANCE Fruit and vegetables consumed raw have become an important vehicle of foodborne illness despite a continuous effort to improve their microbial safety. Salmonella enterica has caused numerous recalls and outbreaks of infection associated with contaminated leafy vegetables. Evidence is increasing that enteric pathogens can reach the leaf apoplast, where they confront plant innate immunity. Plants may be triggered for induction of their defense signaling pathways by exposure to chemical or microbial elicitors. This priming for recognition of microbes by plant defense pathways has been used to inhibit plant pathogens and limit disease. Given that current mitigation strategies are insufficient in preventing microbial contamination of produce and associated outbreaks, we investigated the effect of plant-induced resistance on S. enterica colonization of the lettuce and basil leaf apoplast in order to gain a proof of concept for the use of such an intrinsic approach to inhibit human pathogens in leafy vegetables.

Background: Lettuce-associated respiratory allergy has never been reported before. The aim of this study was to clarify the clinical condition of lettuce-associated respiratory allergy and to identify the lettuce antigen which induces allergic symptoms., Methods: We distributed questionnaires to 1168 lettuce farmers and performed medical examinations in those who exhibited respiratory symptoms related to occupational exposure to lettuce. We analysed specific IgE-binding proteins in the sera of patients through immunoblotting analysis and determined molecular characterization of the IgE-binding bands using liquid chromatography-mass spectrometry., Results: A total of 932 farmers (80%) responded to the questionnaire. Of those, 7% exhibited lettuce-associated respiratory symptoms, during harvesting and packaging. Thirteen patients were diagnosed with allergy to lettuce and agreed to undergo further examinations. The percentage of activated basophils in these patients was significantly higher compared with that reported in negative controls (P < .05). Lettuce-specific IgE (ImmunoCAP ® ) and skin prick testing was positive in 46% and 62% of patients, respectively. Notably, occupational lettuce-allergic asthma was detected in one patient through specific bronchial provocation testing. The IgE-binding bands recognized in the sera of >50% of patients were identified as epidermis-specific secreted glycoprotein EP1-like (51 kDa)., Conclusion: The present analysis identified a novel lettuce allergen. This allergen may have clinically useful applications, such as specific IgE testing and allergen-specific immunotherapy., (© 2020 John Wiley & Sons Ltd.)

Main Conclusion: An edible plant was tested as a host for the production of secretory monoclonal IgA against Shiga toxin 1 (Stx1). The lettuce-derived IgA completely protected Vero cells from Stx1. Secretory immunoglobulin A (SIgA) is thought to control mucosal infections and thus it may be applicable to oral passive immunotherapy. Edible plants are candidate hosts for producing oral formulations with SIgA against pathogenic agents. We previously established a recombinant IgA specific for the B subunit of Shiga toxin 1 (Stx1B) consisting of the Fab fragment of Stx1B-specific monoclonal IgG and the Fc region of IgA (hyIgA). Here, we developed transgenic lettuce (Lactuca sativa) that produces hyIgA in a secretory form (S-hyIgA). An Arabidopsis-derived light-harvesting complex II (LHCB) promoter was used for the expression of all four transgenes (hyIgA heavy, light and j chains, and secretory component). Agrobacterium-mediated transformation was carried out to introduce genes into lettuce leaf discs by means of a single vector harboring all four transgenes. Consistent with the tissue specificity of the LHCB promoter, the expression of hyIgA transgenes was observed in leaf and stem tissues, which contain chloroplasts, at the mRNA and protein levels. The leaves produced hyIgA in a more than tenfold higher yield as compared with stems. The lettuce-derived S-hyIgA was found to bind to Stx1B in a dose-dependent manner by means of ELISA. A leaf extract of the transgenic lettuce completely neutralized the cytotoxicity of Stx1 against Vero cells, which are highly susceptible to Stx1. In conclusion, we established a transgenic lettuce producing a secretory form of hyIgA that can bind bacterial toxin. The results indicate that edible practical plants containing S-hyIgA will provide a possible means for immunotherapy for food poisoning.

Key Message: Two QTLs for resistance to lettuce drop, qLDR1.1 and qLDR5.1, were identified. Associated SNPs will be useful in breeding for lettuce drop and provide the foundation for future molecular analysis. Lettuce drop, caused by Sclerotinia minor and S. sclerotiorum, is an economically important disease of lettuce. The association of resistance to lettuce drop with the commercially undesirable trait of fast bolting has hindered the integration of host resistance in control of this disease. Eruption is a slow-bolting cultivar that exhibits a high level of resistance to lettuce drop. Eruption also is completely resistant to Verticillium wilt caused by race 1 of Verticillium dahliae. A recombinant inbred line population from the cross Reine des Glaces × Eruption was genotyped by sequencing and evaluated for lettuce drop and bolting in separate fields infested with either S. minor or V. dahliae. Two quantitative trait loci (QTLs) for lettuce drop resistance were consistently detected in at least two experiments, and two other QTLs were identified in another experiment; the alleles for resistance at all four QTLs originated from Eruption. A QTL for lettuce drop resistance on linkage group (LG) 5, qLDR5.1, was consistently detected in all experiments and explained 11 to 25% of phenotypic variation. On LG1, qLDR1.1 was detected in two experiments explaining 9 to 12% of the phenotypic variation. Three out of four resistance QTLs are distinct from QTLs for bolting; qLDR5.1 is pleiotropic or closely linked with a QTL for early bolting; however, the rate of bolting shows only a small effect on the variance in resistance observed at this locus. The SNP markers linked with these QTLs will be useful in breeding for resistance through marker-assisted selection.

Background: Verticillium wilt caused by the fungus Verticillium dahliae race 1 is among the top disease concerns for lettuce in the Salinas and Pajaro Valleys of coastal central California. Resistance of lettuce against V. dahliae race 1 was previously mapped to the single dominant Verticillium resistance 1 (Vr1) locus. Lines of tomato resistant to race 1 are known to contain the closely linked Ve1 and Ve2 genes that encode receptor-like proteins with extracellular leucine-rich repeats; the Ve1 and Ve2 proteins act antagonistically to provide resistance against V. dahliae race 1. The Vr1 locus in lettuce contains a cluster of several genes with sequence similarity to the tomato Ve genes. We used genome sequencing and/or PCR screening along with pathogenicity assays of 152 accessions of lettuce to investigate allelic diversity and its relationship to race 1 resistance in lettuce., Results: This approach identified a total of four Ve genes: LsVe1, LsVe2, LsVe3, and LsVe4. The majority of accessions, however, contained a combination of only three of these LsVe genes clustered on chromosomal linkage group 9 (within ~ 25 kb in the resistant cultivar La Brillante and within ~ 127 kb in the susceptible cultivar Salinas)., Conclusions: A single allele, LsVe1L, was present in all resistant accessions and absent in all susceptible accessions. This allele can be used as a molecular marker for V. dahliae race 1 resistance in lettuce. A PCR assay for rapid detection of race 1 resistance in lettuce was designed based on nucleotide polymorphisms. Application of this assay allows identification of resistant genotypes in early stages of plant development or at seed-level without time- and labor-intensive testing in the field.

The concept of "Sweet Immunity" postulates that sugar metabolism and signaling influence plant immune networks. In this study, we tested the potential of commercially available inulin-type fructans to limit disease symptoms caused by Botrytis cinerea in lettuce. Spraying mature lettuce leaves, with inulin-type fructans derived from burdock or chicory was as effective in reducing grey mold disease symptoms caused by Botrytis cinerea as spraying with oligogalacturonides (OGs). OGs are well-known defense elicitors in several plant species. Spraying with inulin and OGs induced accumulation of hydrogen peroxide and levels further increased upon pathogen infection. Inulin and OGs were no longer able to limit Botrytis infection when plants were treated with the ethylene signaling inhibitor 1-methylcyclopropene (1-MCP), indicating that a functional ethylene signaling pathway is needed for the enhanced defense response. Soluble sugars accumulated in leaves primed with OGs, while 1-MCP treatment had an overall negative effect on the sucrose pool. Accumulation of γ-aminobutyric acid (GABA), a stress-associated non-proteinogenic amino acid and possible signaling compound, was observed in inulin-treated samples after infection and negatively affected by the 1-MCP treatment. We have demonstrated for the first time that commercially available inulin-type fructans and OGs can improve the defensive capacity of lettuce, an economically important species. We discuss our results in the context of a possible recognition of fructans as Damage or Microbe Associated Molecular Patterns.

Oral vaccination with edible vaccines is one of the most promising approaches in modern vaccinology. Edible vaccines are an alternative to conventional vaccines, which are typically delivered by injection. Here, freeze-dried transgenic lettuce expressing the cysteine proteinase of the trematode Fasciola hepatica (CPFhW) was used to orally vaccinate cattle and sheep against fasciolosis, which is the most important trematode disease due to the parasite's global distribution, wide spectrum of host species and significant economic losses of farmers. In the study, goals such as reducing the intensity of infection, liver damage and F. hepatica fecundity were achieved. Moreover, we demonstrated that the host sex influenced the outcome of infection following vaccination, with female calves and male lambs showing better protection than their counterparts. Since differences occurred following vaccination and infection, different immunization strategies should be considered for different sexes and host species when developing new control methods. The results of the present study highlight the potential of oral vaccination with plant-made and plant-delivered vaccines for F. hepatica infection control.

Shiga toxin-producing Escherichia coli (STEC) has been associated with illnesses and outbreaks linked to fresh vegetables, prompting a growing public health concern. Most studies regarding interactions of STEC on fresh produce focused on E. coli O157:H7. Limited information is available about survival or fitness of E. coli O104:H4, non-O157 pathogen that was linked to one of the largest outbreaks of hemolytic uremic syndrome in 2011. In this study, survival of E. coli O104:H4 was evaluated on Arabidopsis thaliana plant and lettuce for 5 days compared with E. coli O157:H7, and expression of pathogenesis-realted gene (PR1; induction of plant defense response) was examined by reverse transcription quantitative PCR, and potential influence of capsular polysaccharide (CPS) on the bacterial fitness on plant was investigated. Populations of E. coli O104:H4 strains (RG1, C3493, and LpfA) on Arabidopsis and lettuce were significantly (P < 0.05) greater than those of E. coli O157:H7 strains (7386 and sakai) at day 5 post-inoculation, indicating E. coli O104:H4 may have better survival ability on the plants. In addition, the E. coli O104:H4 strains produced significantly (P < 0.05) higher amounts of CPS compared with the E. coli O157:H7 strains. RG1 strain (1.5-fold) initiated significantly (P < 0.05) lower expression of PR1 gene indicating induction of plant defense response compared with E. coli O157:H7 strains 7386 (2.9-fold) and sakai (2.7-fold). Collectively, the results in this study suggests that different level of CPS production and plant defense response initiated by each STEC strain might influence the bacterial survival or persistence on plants. The present study provides better understanding of survival behavior of STEC, particularly E. coli O104:H4, using a model plant and vegetable under pre-harvest conditions with plant defense response., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

This study aimed to determine the influence of bacterial surface polysaccharides (cellulose, colanic acid, and lipopolysaccharide; LPS) on the colonization or survival of Escherichia coli O157:H7 on plants and the plant defense response. Survival of E. coli O157:H7 were evaluated on Arabidopsis thaliana and romaine lettuce as a model plant and an edible crop (leafy vegetable), respectively. The population of the wild-type strain of E. coli O157:H7 on Arabidopsis plants and lettuce was significantly (P < 0.05) greater compared with the colanic acid-deficient and LPS-truncated mutants on day 1 and day 5 post-inoculation. This result indicates that colanic acid and LPS structures may contribute to the ability of bacterial survival or persistence on plants. The wild-type strain of E. coli O157:H7 produced approximately twice the amount (P < 0.05) of capsular polysaccharide (CPS) than the colanic acid and LPS-truncated mutants. The significantly lower production of CPS was associated with significantly greater (2-fold) expression of pathogenesis-related gene (PR1) compared with the wild-type and cellulose-deficient mutant (P < 0.05). Collectively, the results of this study may suggest that specific surface polysaccharides of E. coli O157:H7 differentially induce the plant defense response, consequently affecting the survival of the human pathogen on plants. The survival and persistence of E. coli O157:H7 was similar on Arabidopsis and lettuce regardless of day post-inoculation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, vomiting, dehydration, weight loss, and high mortality rate in neonatal piglets. Porcine epidemic diarrhea (PED) has been reported in Europe, America, and Asia including Thailand. The disease causes substantial losses to the swine industry in many countries. Presently, there is no effective PEDV vaccine available. In this study, we developed a plant-produced monoclonal antibody (mAb) 2C10 as a prophylactic candidate to prevent the PEDV infection. Recently, plant expression systems have gained interest as an alternative for the production of antibodies because of many advantages, such as low production cost, lack of human and animal pathogen, large scalability, etc. The 2C10 mAb was transiently expressed in Nicotiana benthamiana and lettuce using geminiviral vector. After purification by protein A affinity chromatography, the antibody was tested for the binding and neutralizing activity against PEDV. Our result showed that the plant produced 2C10 mAb can bind to the virus and also inhibit PEDV infection in vitro . These results show excellent potential for a plant-expressed 2C10 as a PEDV prophylaxis and a diagnostic for PEDV infection., Competing Interests: Conflict of Interest: On behalf of my co-authors, I declare that there are no financial or other conflicts of interest in the present manuscript. This work was original research, and the data presented in this article have not been published in elsewhere., (Georg Thieme Verlag KG Stuttgart · New York.)

Human norovirus is the leading cause of foodborne illness worldwide with the majority of outbreaks linked to fresh produce and leafy greens. It is essential that we thoroughly understand the type of relationship and interactions that take place between plants and human norovirus to better utilize control strategies to reduce transmission of norovirus in the field onto plants harvested for human consumption. In this study the expression of gene markers for the salicylic acid (SA) and jasmonic acid (JA) plant defense pathways was measured and compared in romaine lettuce (Lactuca sativa) and Arabidopsis thaliana Col-0 plants that were inoculated with Murine Norovirus-1, Tulane Virus, human norovirus GII.4, or Hank's Balanced Salt Solution (control). Genes involving both the SA and JA pathways were expressed in both romaine lettuce and A. thaliana for all three viruses, as well as controls. Studies, including gene expression of SA- and JA-deficient A. thaliana mutant lines, suggest that the JA pathway is more likely involved in the plant immune response to human norovirus. This research provides the first pieces of information regarding how foodborne viruses interact with plants in the preharvest environment.

Background and Objective: Lipid transfer protein (LTP) sensitization is the most common cause of food allergy in the Mediterranean area, with peach allergy acting as the primary sensitizer in most cases. Lettuce has been described as a common offending food in patients with LTP syndrome. The aim of the study was to investigate the frequency and clinical expression of LTP syndrome in a sample of lettuceallergic patients., Methods: We determined specific IgE to Pru p 3 and lettuce in a sample of 30 patients with a diagnosis of lettuce allergy. Symptoms elicited by other LTP-containing plant-derived foods and the presence of cofactors were assessed., Results: The clinical symptoms of lettuce allergy were frequently severe, with 18 of the 30 patients experiencing anaphylaxis. All the patients had allergic reactions to other plant foods. Cofactors were involved in the clinical reactions of 13 of the 30 patients. Sensitization to pollens was found in 90% of patients., Conclusions: Lettuce allergy is found not as an isolated condition but in the context of LTP syndrome and it is characterized by severe reactions and frequent cofactor association.

Lettuce (Lactuca sativa L.) is a diploid (2n = 18) with a genome size of 2,600 Mbp, and belongs to the family Compositae. Bacterial leaf spot (BLS), caused by Xanthomonas campestris pv. vitians, is a major disease of lettuce worldwide. Leaf lettuce PI 358001-1 has been characterized as an accession highly resistant to BLS and has white seed. In order to understand inheritance of the high resistance in this germplasm line, an F 3 population consisting of 163 families was developed from the cross PI 358001-1 × 'Tall Guzmaine' (a susceptible Romaine lettuce variety with black seed). The segregation ratio of reaction to disease by seedling inoculation with X. campestris pv. vitians L7 strain in the F 3 families was shown to be 32:82:48 homozygous resistant/heterozygous/homozygous susceptible, fitting to 1:2:1 (n = 162, χ 2 = 3.19, P = 0.20). The segregation ratio of seed color by checking F 2 plants was 122:41 black/white, fitting to 3:1 (n = 163, χ 2 = 0.002, P = 0.96). The results indicated that both BLS resistance and seed color were inherited as a dominant gene mode. A genetic linkage map based on 124 randomly selected F 2 plants was developed to enable molecular mapping of the BLS resistance and the seed color trait. In total, 199 markers, comprising 176 amplified fragment length polymorphisms, 16 simple-sequence repeats, 5 resistant gene candidate markers, and 2 cleaved amplified polymorphic sequences (CAPS) markers were assigned to six linkage groups. The dominant resistance gene to BLS (Xcvr) was mapped on linkage group 2 and the gene locus y for seed color was identified on linkage group 5. Due to the nature of a single gene inheritance, the high-resistance gene should be readily transferred to adapted lettuce cultivars to battle against the devastating disease of lettuce.

The commercially available inoculant Bacillus amyloliquefaciens FZB42 is able to considerably reduce lettuce bottom rot caused by Rhizoctonia solani. To understand the interaction between FZB42 and R. solani in the rhizosphere of lettuce, we used an axenic system with lettuce bacterized with FZB42 and inoculated with R. solani. Confocal laser scanning microscopy showed that FZB42 could delay the initial establishment of R. solani on the plants. To show which secondary metabolites of FZB42 are produced under these in-situ conditions, we developed an ultra-high performance liquid chromatography coupled to time of flight mass spectrometry-based method and identified surfactin, fengycin, and bacillomycin D in the lettuce rhizosphere. We hypothesized that lipopeptides and polyketides play a role in enhancing the plant defense responses in addition to the direct antagonistic effect toward R. solani and used a quantitative real-time polymerase chain reaction-based assay for marker genes involved in defense signaling pathways in lettuce. A significant higher expression of PDF 1.2 observed in the bacterized plants in response to subsequent pathogen challenge showed that FZB42 could enhance the lettuce defense response toward the fungal pathogen. To identify if surfactin or other nonribosomally synthesized secondary metabolites could elicit the observed enhanced defense gene expression, we examined two mutants of FZB42 deficient in production of surfactin and the lipopetides and polyketides, by expression analysis and pot experiments. In the absence of surfactin and other nonribosomally synthesized secondary metabolites, there was no enhanced PDF 1.2-mediated response to the pathogen challenge. Pot experiment results showed that the mutants failed to reduce disease incidence in lettuce as compared with the FZB42 wild type, indicating, that surfactin as well as other nonribosomally synthesized secondary metabolites play a role in the actual disease suppression and on lettuce health. In conclusion, our study showed that nonribosomally synthesized secondary metabolites of FZB42 are actually produced in the lettuce rhizosphere and contribute to the disease suppression by mediating plant defense gene expression toward the pathogen R. solani.

Many cultivars of lettuce (Lactuca sativa L.) are susceptible to downy mildew, a nearly globally ubiquitous disease caused by Bremia lactucae. We previously determined that Batavia type cultivar 'La Brillante' has a high level of field resistance to the disease in California. Testing of a mapping population developed from a cross between 'Salinas 88' and La Brillante in multiple field and laboratory experiments revealed that at least five loci conferred resistance in La Brillante. The presence of a new dominant resistance gene (designated Dm50) that confers complete resistance to specific isolates was detected in laboratory tests of seedlings inoculated with multiple diverse isolates. Dm50 is located in the major resistance cluster on linkage group 2 that contains at least eight major, dominant Dm genes conferring resistance to downy mildew. However, this Dm gene is ineffective against the isolates of B. lactucae prevalent in the field in California and the Netherlands. A quantitative trait locus (QTL) located at the Dm50 chromosomal region (qDM2.2) was detected, though, when the amount of disease was evaluated a month before plants reached harvest maturity. Four additional QTL for resistance to B. lactucae were identified on linkage groups 4 (qDM4.1 and qDM4.2), 7 (qDM7.1), and 9 (qDM9.2). The largest effect was associated with qDM7.1 (up to 32.9% of the total phenotypic variance) that determined resistance in multiple field experiments. Markers identified in the present study will facilitate introduction of these resistance loci into commercial cultivars of lettuce.

Phylogeographic studies inform about routes of pathogen dissemination and are instrumental for improving import/export controls. Genomes of 17 isolates of the bacterial wilt and potato brown rot pathogen Ralstonia solanacearum race 3 biovar 2 (R3bv2), a Select Agent in the United States, were thus analyzed to get insight into the phylogeography of this pathogen. Thirteen of fourteen isolates from Europe, Africa, and Asia were found to belong to a single clonal lineage while isolates from South America were genetically diverse and tended to carry ancestral alleles at the analyzed genomic loci consistent with a South American origin of R3bv2. The R3bv2 isolates share a core repertoire of 31 type III-secreted effector genes representing excellent candidates to be targeted with resistance genes in breeding programs to develop durable disease resistance. Toward this goal, 27 R3bv2 effectors were tested in eggplant, tomato, pepper, tobacco, and lettuce for induction of a hypersensitive-like response indicative of recognition by cognate resistance receptors. Fifteen effectors, eight of them core effectors, triggered a response in one or more plant species. These genotypes may harbor resistance genes that could be identified and mapped, cloned, and expressed in tomato or potato, for which sources of genetic resistance to R3bv2 are extremely limited.

We previously showed that allelic genes mol¹ and mo1² used to protect lettuce crops against Lettuce mosaic virus (LMV) correspond to mutant alleles of the gene encoding the eukaryotic translation initiation factor 4E. LMV resistance-breaking determinants map not only to the main potyvirus virulence determinant, a genome-linked viral protein, but also to the C-terminal region of the cylindrical inclusion (CI), with a key role of amino acid at position 621. Here, we show that the propagation of several non-lettuce isolates of LMV in mo1¹ plants is accompanied by a gain of virulence correlated with the presence in the CI C terminus of a serine at position 617 and the accumulation of mutations at positions 602 or 627. Whole-genome sequencing of native and evolved isolates showed that no other mutation could be associated with adaptation to mo1 resistance. Site-directed mutagenesis pinpointed the key role in the virulence of the combination of mutations at positions 602 and 617, in addition to position 621. The impact of these mutations on the fitness of the virus was evaluated, suggesting that the durability of mo1 resistance in the field relies on the fitness cost associated with the resistance-breaking mutations, the nature of the mutations, and their potential antagonistic effects.

Key Message: In a stacking study of eight resistance QTLs in lettuce against downy mildew, only three out of ten double combinations showed an increased resistance effect under field conditions. Complete race nonspecific resistance to lettuce downy mildew, as observed for the nonhost wild lettuce species Lactuca saligna, is desired in lettuce cultivation. Genetic dissection of L. saligna's complete resistance has revealed several quantitative loci (QTL) for resistance with field infection reductions of 30-50 %. To test the effect of stacking these QTL, we analyzed interactions between homozygous L. saligna CGN05271 chromosome segments introgressed into the genetic background of L. sativa cv. Olof. Eight different backcross inbred lines (BILs) with single introgressions of 30-70 cM and selected predominately for quantitative resistance in field situations were intercrossed. Ten developed homozygous lines with stacked introgression segments (double combinations) were evaluated for resistance in the field. Seven double combinations showed a similar infection as the individual most resistant parental BIL, revealing epistatic interactions with 'less-than-additive' effects. Three double combinations showed an increased resistance level compared to their parental BILs and their interactions were additive, 'less-than-additive' epistatic and 'more-than-additive' epistatic, respectively. The additive interaction reduced field infection by 73 %. The double combination with a 'more-than-additive' epistatic effect, derived from a combination between a susceptible and a resistant BIL with 0 and 30 % infection reduction, respectively, showed an average field infection reduction of 52 %. For the latter line, an attempt to genetically dissect its underlying epistatic loci by substitution mapping did not result in smaller mapping intervals as none of the 22 substitution lines reached a similar high resistance level. Implications for breeding and the inheritance of L. saligna's complete resistance are discussed.

Fusarium wilts of leafy vegetables are difficult to manage under intensive cropping systems. The objective of this study was to evaluate, in three experimental trials, the susceptibility of commercial cultivars of lettuce, wild and cultivated rocket and lamb's lettuce to Fusarium wilts in orderto provide information to breeders, as well as to growers. Some of the cultivars of lettuce tested were completely resistant to the three races of Fusarium wilt. It is interesting to observe that most of the resistant cultivars were 'Batavia red'. Only few rocket cultivars commercially available show a partial resistant reaction to F. oxysporum f.sp. raphani, while, varietal resistance is not applicable at the moment to control Fusarium wilt of lamb's lettuce. The integration of cultural practices, use of resistant cultivars, when available, chemicals and biological control agents, permit to prevent and manage these important diseases on leafy vegetables for fresh-cut production.

Key Message: Three regions with quantitative resistance to downy mildew of non-host and wild lettuce species, Lactuca saligna , disintegrate into seventeen sub-QTLs with plant-stage-dependent effects, reducing or even promoting the infection. Previous studies on the genetic dissection of the complete resistance of wild lettuce, Lactuca saligna, to downy mildew revealed 15 introgression regions that conferred plant stage dependent quantitative resistances (QTLs). Three backcross inbred lines (BILs), carrying an individual 30-50 cM long introgression segment from L. saligna in a cultivated lettuce, L. sativa, background, reduced infection by 60-70 % at young plant stage and by 30-50 % at adult plant stage in field situations. We studied these three quantitative resistances in order to narrow down their mapping interval and determine their number of loci, either single or multiple. We performed recombinant screenings and developed near isogenic lines (NILs) with smaller overlapping L. saligna introgressions (substitution mapping). In segregating introgression line populations, recombination was suppressed up to 17-fold compared to the original L. saligna × L. sativa F 2 population. Recombination suppression depended on the chromosome region and was stronger suppressed at the smallest introgression lengths. Disease evaluation of the NILs revealed that the resistance of all three BILs was not explained by a single locus but by multiple sub-QTLs. The 17 L. saligna-derived sub-QTLs had a smaller and plant stage dependent resistance effect, some segments reducing; others even promoting downy mildew infection. Implications for lettuce breeding are outlined.

Scope: Today, about 2-8% of the population of Western countries exhibits some type of food allergy whose impact ranges from localized symptoms confined to the oral mucosa to severe anaphylactic reactions. Consumed worldwide, lettuce is a Compositae family vegetable that can elicit allergic reactions. To date, however, only one lipid transfer protein has been described in allergic reaction to lettuce. The aim of this study was to identify potential new allergens involved in lettuce allergy., Methods and Results: Sera from 42 Spanish lettuce-allergic patients were obtained from patients recruited at the outpatient clinic. IgE-binding proteins were detected by SDS-PAGE and immunoblotting. Molecular characterization of IgE-binding bands was performed by MS. Thaumatin was purified using the Agilent 3100 OFFGEL system. The IgE-binding bands recognized in the sera of more than 50% of patients were identified as lipid transfer protein (9 kDa), a thaumatin-like protein (26 kDa), and an aspartyl protease (35 and 45 kDa). ELISA inhibition studies were performed to confirm the IgE reactivity of the purified allergen., Conclusion: Two new major lettuce allergens-a thaumatin-like protein and an aspartyl protease-have been identified and characterized. These allergens may be used to improve both diagnosis and treatment of lettuce-allergic patients., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Breeding lettuce (Lactuca sativa) for resistance to the downy mildew pathogen Bremia lactucae is mainly achieved by introgression of dominant downy mildew resistance (Dm) genes. New Bremia races quickly render Dm genes ineffective, possibly by mutation of recognized host-translocated effectors or by suppression of effector-triggered immunity. We have previously identified 34 potential RXLR(-like) effector proteins of B. lactucae that were here tested for specific recognition within a collection of 129 B. lactucae-resistant Lactuca lines. Two effectors triggered a hypersensitive response: BLG01 in 52 lines, predominantly L. saligna, and BLG03 in two L. sativa lines containing Dm2 resistance. The N-terminal sequences of BLG01 and BLG03, containing the signal peptide and GKLR variant of the RXLR translocation motif, are not required for in planta recognition but function in effector delivery. The locus responsible for BLG01 recognition maps to the bottom of lettuce chromosome 9, whereas recognition of BLG03 maps in the RGC2 cluster on chromosome 2. Lactuca lines that recognize the BLG effectors are not resistant to Bremia isolate Bl:24 that expresses both BLG genes, suggesting that Bl:24 can suppress the triggered immune responses. In contrast, lettuce segregants displaying Dm2-mediated resistance to Bremia isolate Bl:5 are responsive to BLG03, suggesting that BLG03 is a candidate Avr2 protein.

Many cultivars of lettuce (Lactuca sativa L.), the most popular leafy vegetable, are susceptible to downy mildew disease caused by Bremia lactucae. Cultivars Iceberg and Grand Rapids that were released in the 18th and 19th centuries, respectively, have high levels of quantitative resistance to downy mildew. We developed a population of recombinant inbred lines (RILs) originating from a cross between these two legacy cultivars, constructed a linkage map, and identified two QTLs for resistance on linkage groups 2 (qDM2.1) and 5 (qDM5.1) that determined resistance under field conditions in California and the Netherlands. The same QTLs determined delayed sporulation at the seedling stage in laboratory experiments. Alleles conferring elevated resistance at both QTLs originate from cultivar Iceberg. An additional QTL on linkage group 9 (qDM9.1) was detected through simultaneous analysis of all experiments with mixed-model approach. Alleles for elevated resistance at this locus originate from cultivar Grand Rapids.

Consumption of fresh produce contaminated with bacterial human pathogens has resulted in various, sometimes deadly, disease outbreaks. In this study, we assessed plant defense responses induced by the fully pathogenic bacteria Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium SL1344 in both Arabidopsis thaliana and lettuce (Lactuca sativa). Unlike SL1344, O157:H7 induced strong plant immunity at both pre-invasion and post-invasion steps of infection. For instance, O157:H7 triggered stomatal closure even under high relative humidity, an environmental condition that generally weakens plant defenses against bacteria in the field and laboratory conditions. SL1344 instead induced a transient stomatal immunity. We also observed that PR1 gene expression was significantly higher in Arabidopsis leaves infected with O157:H7 compared with SL1344. These results suggest that plants may recognize and respond to some human pathogens more effectively than others. Furthermore, stomatal immunity can diminish the penetration of human pathogens through the leaf epidermis, resulting in low bacterial titers in the plant apoplast and suggesting that additional control measures can be employed to prevent food contamination. The understanding of how plant responses can diminish bacterial contamination is paramount in preventing outbreaks and improving the safety of food supplies.

Tuberculosis (TB) caused by Mycobacterium tuberculosis is one of the leading fatal infectious diseases. The development of TB vaccines has been recognized as a major public health priority by the World Health Organization. In this study, three candidate antigens, ESAT-6 (6 kDa early secretory antigenic target) and Mtb72F (a fusion polyprotein from two TB antigens, Mtb32 and Mtb39) fused with cholera toxin B-subunit (CTB) and LipY (a cell wall protein) were expressed in tobacco and/or lettuce chloroplasts to facilitate bioencapsulation/oral delivery. Site-specific transgene integration into the chloroplast genome was confirmed by Southern blot analysis. In transplastomic leaves, CTB fusion proteins existed in soluble monomeric or multimeric forms of expected sizes and their expression levels varied depending upon the developmental stage and time of leaf harvest, with the highest-level of accumulation in mature leaves harvested at 6PM. The CTB-ESAT6 and CTB-Mtb72F expression levels reached up to 7.5% and 1.2% of total soluble protein respectively in mature tobacco leaves. Transplastomic CTB-ESAT6 lettuce plants accumulated up to 0.75% of total leaf protein. Western blot analysis of lyophilized lettuce leaves stored at room temperature for up to six months showed that the CTB-ESAT6 fusion protein was stable and preserved proper folding, disulfide bonds and assembly into pentamers for prolonged periods. Also, antigen concentration per gram of leaf tissue was increased 22 fold after lyophilization. Hemolysis assay with purified CTB-ESAT6 protein showed partial hemolysis of red blood cells and confirmed functionality of the ESAT-6 antigen. GM1-binding assay demonstrated that the CTB-ESAT6 fusion protein formed pentamers to bind with the GM1-ganglioside receptor. The expression of functional Mycobacterium tuberculosis antigens in transplastomic plants should facilitate development of a cost-effective and orally deliverable TB booster vaccine with potential for long-term storage at room temperature. To our knowledge, this is the first report of expression of TB vaccine antigens in chloroplasts.

Noroviruses are the leading cause of food-borne outbreaks, including those that involve lettuce. The culturable porcine sapovirus (SaV) was used as a norovirus surrogate to study the persistence and the potential transfer of the virus from roots to leaves and from outer to inner leaves of lettuce plants. Treatment of lettuce with SaV was done through the roots of young plants, the soil, or the outer leaves of mature plants. Sampling of roots, xylem sap, and inner and outer leaves followed by RNA extraction and SaV-specific real-time reverse transcription (RT)-PCR was performed at 2 h and on postinoculation days (PID) 2, 5, 7, 14, and/or 28. When SaV was inoculated through the roots, viral RNA persisted on the roots and in the leaves until PID 28. When the virus was inoculated through the soil, viral RNA was detected on the roots and in the xylem sap until PID 14; viral RNA was detected in the leaves only until PID 2. No infectious virus was detected inside the leaves for either treatment. When SaV was inoculated through the outer leaves, viral RNA persisted on the leaves until PID 14; however, the virus did not transfer to inner leaves. Infectious viral particles on leaves were detected only at 2 h postinoculation. The milky sap (latex) of leaves, but not the roots' xylem sap, significantly decreased virus infectivity when tested in vitro. Collectively, our results showed the transfer of SaV from roots to leaves through the xylem system and the capacity of the sap of lettuce leaves to decrease virus infectivity in leaves.

Efficient immunization against hepatitis B virus (HBV) and other pathogens with plant-based oral vaccines requires appropriate plant expressors and the optimization of vaccine compositions and administration protocols. Previous immunization studies were mainly based on a combination of the injection of a small surface antigen of HBV (S-HBsAg) and the feeding with raw tissue containing the antigen, supplemented with an adjuvant, and coming from plants conferring resistance to kanamycin. The objective of this study was to develop a prototype oral vaccine formula suitable for human immunization. Herbicide-resistant lettuce was engineered, stably expressing through progeny generation micrograms of S-HBsAg per g of fresh weight and formed into virus-like particles (VLPs). Lyophilized tissue containing a relatively low, 100-ng VLP-assembled antigen dose, administered only orally to mice with a long, 60-day interval between prime and boost immunizations and without exogenous adjuvant, elicited mucosal and systemic humoral anti-HBs responses at the nominally protective level. Lyophilized tissue was converted into tablets, which preserved S-HBsAg content for at least one year of room temperature storage. The results of the study provide indications on immunization methodology using a durable, efficacious, and convenient plant-derived prototype oral vaccine against hepatitis B.

Cholera and malaria are major diseases causing high mortality. The only licensed cholera vaccine is expensive; immunity is lost in children within 3 years and adults are not fully protected. No vaccine is yet available for malaria. Therefore, in this study, the cholera toxin-B subunit (CTB) of Vibrio cholerae fused to malarial vaccine antigens apical membrane antigen-1 (AMA1) and merozoite surface protein-1 (MSP1) was expressed in lettuce and tobacco chloroplasts. Southern blot analysis confirmed homoplasmy and stable integration of transgenes. CTB-AMA1 and CTB-MSP1 fusion proteins accumulated up to 13.17% and 10.11% (total soluble protein, TSP) in tobacco and up to 7.3% and 6.1% (TSP) in lettuce, respectively. Nine groups of mice (n = 10/group) were immunized subcutaneously (SQV) or orally (ORV) with purified antigens or transplastomic tobacco leaves. Significant levels of antigen-specific antibody titres of immunized mice completely inhibited proliferation of the malarial parasite and cross-reacted with the native parasite proteins in immunoblots and immunofluorescence studies. Protection against cholera toxin challenge in both ORV (100%) and SQV (89%) mice correlated with CTB-specific titres of intestinal, serum IgA and IgG1 in ORV and only IgG1 in SQV mice, but no other immunoglobulin. Increasing numbers of interleukin-10(+) T cell but not Foxp3(+) regulatory T cells, suppression of interferon-gamma and absence of interleukin-17 were observed in protected mice, suggesting that immunity is conferred via the Tr1/Th2 immune response. Dual immunity against two major infectious diseases provided by chloroplast-derived vaccine antigens for long-term (>300 days, 50% of mouse life span) offers a realistic platform for low cost vaccines and insight into mucosal and systemic immunity.

The obtainment of transgenic edible plants carrying recombinant antigens is a desired issue in search for economic alternatives viewing vaccine production. Here we report a strategy for genetic transformation of lettuce plants (Lactuca sativa L.) using the surface antigen HBsAg of hepatitis B virus. Transgenic lettuce seedlings were obtained through the application of a regulated balance of plant growth regulators. Genetic transformation process was acquired by cocultivation of cotyledons with Agrobacterium tumefaciens harboring the recombinant plasmid. It is the first description of a lettuce Brazilian variety 'Vitória de Verão' genetically modified.

Background: IgE sensitisation to non-specific lipid transfer proteins (nsLTP), e.g., Pru p 3 the major allergen from peach and most important allergenic LTP, is strongly associated with severe symptoms in food allergic patients. Lac s 1, a member of the nsLTP protein family, was recently identified as major allergen in lettuce (Lactuca sativa), but has not yet been investigated on the molecular basis., Objective: Molecular characterisation and immunological comparison of Lac s 1 to peach allergen Pru p 3., Methods: Lac s 1 cDNA was cloned by RT-PCR and natural (n) Lac s 1 was purified by a two-step chromatography. Protein structure was verified by N-terminal sequencing, mass spectrometry, and circular dichroism spectroscopy. Immunoblotting, ImmunoCAP, and competitive IgE binding experiments were performed to study the IgE sensitisation pattern and cross-reactivity with Pru p 3. Allergenic potency was analysed by histamine release assay., Results: Twenty-nine lettuce allergic patients, with or without concomitant peach allergy, and 19 peach allergic patients without lettuce allergy were included in this study. IgE reactivity to lettuce was due to mono-sensitisation to Lac s 1 or cross-reactive glycan structures. Two Lac s 1 isoforms were identified which showed amino acid identity (aa-id) of 62% to each other, up to 66% to Pru p 3, and 72% to the N-terminal peptide of plane pollen LTP Pla a 3. The prevalence of IgE binding to nLac s 1 was 90% using lettuce extract in immunoblotting experiments. Enhanced sensitivity was observed in ImmunoCAP using purified nLac s 1 in comparison to extracts (93% versus 76%). Although IgE sensitisation to Lac s 1 and Pru p 3 was strongly associated, the two LTPs showed different IgE binding properties. Sensitisation to LTPs does not necessarily reflect the clinical disease, but Lac s 1 was capable of triggering histamine release as shown by positive skin test results in Lac s 1 mono-sensitised patients and by in vitro mediator release assays., Conclusion: Purified nLac s 1 will enhance the sensitivity in component resolved diagnosis of lettuce allergy. Similar to other cross-reactive food allergies, exclusive testing of IgE reactivities to LTP cannot be used as biomarker for clinical relevance. Our data provide indirect evidence that Pru p 3 might act as the primary sensitising agent in patients allergic to both lettuce and peach.

Resistance genes can exhibit heterogeneous patterns of variation. However, there are few data on their frequency and variation in natural populations. We analysed the frequency and variation of the resistance gene Dm3, which confers resistance to Bremia lactucae (downy mildew) in 1033 accessions of Lactuca serriola (prickly lettuce) from 49 natural populations. Inoculations with an isolate of Bremia lactucae carrying avirulence gene Avr3 indicated that the frequency of Dm3 in natural populations of L. serriola was very low. Molecular analysis demonstrated that Dm3 was present in only one of the 1033 wild accessions analysed. The sequence of the 5' region of Dm3 was either highly conserved among accessions, or absent. In contrast, frequent chimeras were detected in the 3' leucine-rich repeat-encoding region. Therefore low frequency of the Dm3 specificity in natural populations was due to either the recent evolution of Dm3 specificity, or deletions of the whole gene as well as variation in 3' region caused by frequent gene conversions. This is the most extensive analysis of the prevalence of a known disease resistance gene to date, and indicates that the total number of resistance genes in a species may be very high. This has implications for the scales of germplasm conservation and exploitation of sources of resistance.

The eIF4E and eIF(iso)4E cDNAs from several genotypes of lettuce (Lactuca sativa) that are susceptible, tolerant, or resistant to infection by Lettuce mosaic virus (LMV; genus Potyvirus) were cloned and sequenced. Although Ls-eIF(iso)4E was monomorphic in sequence, three types of Ls-eIF4E differed by point sequence variations, and a short in-frame deletion in one of them. The amino acid variations specific to Ls-eIF4E(1) and Ls-eIF4E(2) were predicted to be located near the cap recognition pocket in a homology-based tridimensional protein model. In 19 lettuce genotypes, including two near-isogenic pairs, there was a strict correlation between these three allelic types and the presence or absence of the recessive LMV resistance genes mo1(1) and mo1(2). Ls-eIF4E(1) and mo1(1) cosegregated in the progeny of two separate crosses between susceptible genotypes and an mo1(1) genotype. Finally, transient ectopic expression of Ls-eIF4E restored systemic accumulation of a green fluorescent protein-tagged LMV in LMV-resistant mo1(2) plants and a recombinant LMV expressing Ls-eIF4E degrees from its genome, but not Ls-eIF4E(1) or Ls-eIF(iso)4E, accumulated and produced symptoms in mo1(1) or mo1(2) genotypes. Therefore, sequence correlation, tight genetic linkage, and functional complementation strongly suggest that eIF4E plays a role in the LMV cycle in lettuce and that mo1(1) and mo1(2) are alleles coding for forms of eIF4E unable or less effective to fulfill this role. More generally, the isoforms of eIF4E appear to be host factors involved in the cycle of potyviruses in plants, probably through a general mechanism yet to be clarified.

Background: Allergic reactions to lettuce (Lactuca sativa) are not too frequent and few cases of systemic adverse reactions after its ingestion have been described., Objective: We report a case of clinical sensitization to lettuce on a patient who presented mucocutaneous manifestations after its ingestion, with positive skin tests, histamine release test and serum specific-IgE to lettuce. The allergens responsible for this sensitization were also characterized by means of SDS-PAGE immunoblotting., Materials and Methods: We performed skin tests, histamine release test, serum specific IgE determination and CAP inhibition with lettuce and mugwort (Artemisia vulgaris) extracts. An aqueous and enriched lettuce (from loose leaf type) extract was subjected to SDS-PAGE immunoblotting for determination of its IgE-binding components., Results and Conclusions: CAP inhibition showed antigenic community between lettuce and mugwort. Four protein bands from the lettuce extracts with molecular weights of 50, 43, 39 and 16 kDa exhibited IgE-binding properties.

Background: Food allergies are usually recognized by historical triggers; yet, on occasion, objective testing may be needed to demonstrate allergic causes more convincingly., Objective: Two atopic adult patients presented with respiratory and/or ocular symptoms with an unusual association to handling or eating of vegetables. A third atopic patient presented without attribution of symptoms to vegetable exposure. In all, work-up had revealed positive skin tests and in vitro assays for specific IgE to carrot and/or lettuce; none had gastrointestinal complaints., Methods: To assess further these unusual histories and/or IgE antibody findings, laboratory challenge was performed with nasal resistance being used as a monitor of rhinitic reaction., Results: The first two patients had prolonged nasal objective responses to challenge; the third had a transient response to one of two challenges., Conclusion: We conclude that lettuce and carrot can account for clinically significant IgE-mediated allergic reactions, manifested by nasal obstruction. This laboratory reaction to foodstuffs appears to correlate with patient-reported upper airway and ocular symptoms.