A Robust Functional Genomics Approach to Identify Effector Genes Required for Thrips (Frankliniella occidentalis) Reproductive Performance on Tomato Leaf Discs

Thrips (Frankliniella occidentalis) is a persistent plant pest that is able to vector pathogenic viruses. Natural plant resistance to thrips has become a prominent breeding target in commercial crops. The main reason for this is the shift toward banning key pesticides used for controlling thrips infestations and the lack of effective alternatives. Despite this urgent need for crop plants that are resistant, or tolerant, to thrips infestation, the toolbox for studying genetic resistance to this insect is still underdeveloped. Essentially, there is a lack of robust protocols for the screening and identification of thrips genes relevant to its performance on crop plants. To bridge this gap, we have developed a functional analysis screening method. Our approach relies on the, Agrobacterium tumefaciens-mediated, homogeneous, and transient ectopic expression of thrips genes in large tomato leaf discs followed by the assessment of thrips reproductive performance. The setup is designed to maintain gene expression during the course of the assay, where GFP signal in the control treatment is used as a reporter of expression. The screen is conducted in a climate box under controlled settings. As a result, multiple genes can be screened for their effect on thrips reproductive performance in a single experiment and in a relatively small space, without the need for generating stable transgenic plants. The method also eliminates the need for a greenhouse equipped to accommodate the combination of A. tumefaciens-infiltrations and thrips infestations. It is not only flexible and convenient for screening genes encoding putative thrips effectors but also for plant resistance genes or effector-targets of host plants and can be adapted for other crop plants, or other herbivorous arthropods

High-density SNP-based association mapping of seed traits in fenugreek reveals homology with clover

Fenugreek as a self-pollinated plant is ideal for genome-wide association mapping where traits can be marked by their association with natural mutations. However, fenugreek is poorly investigated at the genomic level due to the lack of information regarding its genome. To fill this gap, we genotyped a collection of 112 genotypes with 153,881 SNPs using double digest restriction site-associated DNA sequencing. We used 38,142 polymorphic SNPs to prove the suitability of the population for association mapping. One significant SNP was associated with both seed length and seed width, and another SNP was associated with seed color. Due to the lack of a comprehensive genetic map, it is neither possible to align the newly developed markers to chromosomes nor to predict the underlying genes. Therefore, systematic targeting of those markers to homologous genomes of other legumes can overcome those problems. A BLAST search using the genomic fenugreek sequence flanking the identified SNPs showed high homology with several members of the Trifolieae tribe indicating the potential of translational approaches to improving our understanding of the fenugreek genome. Using such a comprehensively-genotyped fenugreek population is the first step towards identifying genes underlying complex traits and to underpin fenugreek marker-assisted breeding programs

The tomato phosphatidylinositol-phospholipase C2 (SlPLC2) is required for defense gene induction by the fungal elicitor xylanase

The tomato [Solanum lycopersicum (Sl)] phosphatidylinositol-phospholipase C (PI-PLC) gene family is composed of six members, named SlPLC1 to SlPLC6, differentially regulated upon pathogen attack. We have previously shown that the fungal elicitor xylanase rapidly induces nitric oxide (NO), which is required for PI-PLCs activity and downstream defense responses in tomato cell suspensions. Here, we show that all six SlPLC genes are expressed in tomato cell suspensions. Treatment of the cells with xylanase induces an early increase in SlPLC5 transcript levels, followed by a raise of the amount of SlPLC2 transcripts. The production of NO is required to augment SlPLC5 transcript levels in xylanase-treated tomato cells. Xylanase also induces SlPLC2 and SlPLC5 transcript levels in planta. We knocked-down the expression of SlPLC2 and SlPLC5 by virus-induced gene silencing. We found that SlPLC2 is required for xylanase-induced expression of the defense-related genes PR1 and HSR203J.Fil: Gonorazky, Ana Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Ramírez, Leonor. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Abd El Haliem, Ahmed. Wageningen University; Países BajosFil: Vossen, Jack H.. Wageningen University; Países BajosFil: Lamattina, Lorenzo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Ten Have, Arjen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Joosten, Matthieu H. A. J.. Wageningen University; Países BajosFil: Laxalt, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentin

NLR network mediates immunity to diverse plant pathogens

Both plants and animals rely on nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins to respond to invading pathogens and activate immune responses. An emerging concept of NLR function is that “sensor” NLR proteins are paired with “helper” NLRs to mediate immune signaling. However, our fundamental knowledge of sensor/helper NLRs in plants remains limited. In this study, we discovered a complex NLR immune network in which helper NLRs in the NRC (NLR required for cell death) family are functionally redundant but display distinct specificities toward different sensor NLRs that confer immunity to oomycetes, bacteria, viruses, nematodes, and insects. The helper NLR NRC4 is required for the function of several sensor NLRs, including Rpi-blb2, Mi-1.2, and R1, whereas NRC2 and NRC3 are required for the function of the sensor NLR Prf. Interestingly, NRC2, NRC3, and NRC4 redundantly contribute to the immunity mediated by other sensor NLRs, including Rx, Bs2, R8, and Sw5. NRC family and NRC-dependent NLRs are phylogenetically related and cluster into a well-supported superclade. Using extensive phylogenetic analysis, we discovered that the NRC superclade probably emerged over 100 Mya from an NLR pair that diversified to constitute up to one-half of the NLRs of asterids. These findings reveal a complex genetic network of NLRs and point to a link between evolutionary history and the mechanism of immune signaling. We propose that this NLR network increases the robustness of immune signaling to counteract rapidly evolving plant pathogens

Allelic variants of the NLR protein Rpi-chc1 differentially recognize members of the Phytophthora infestans PexRD12/31 effector superfamily through the leucine-rich repeat domain

Phytophthora infestans is a pathogenic oomycete that causes the infamous potato late blight disease. Resistance (R) genes from diverse Solanum species encode intracellular receptors that trigger effective defense responses upon the recognition of cognate RXLR avirulence (Avr) effector proteins. To deploy these R genes in a durable fashion in agriculture, we need to understand the mechanism of effector recognition and the way the pathogen evades recognition. In this study, we cloned 16 allelic variants of the Rpi-chc1 gene from Solanum chacoense and other Solanum species, and identified the cognate P. infestans RXLR effectors. These tools were used to study effector recognition and co-evolution. Functional and non-functional alleles of Rpi-chc1 encode coiled-coil nucleotide-binding leucine-rich repeat (CNL) proteins, being the first described representatives of the CNL16 family. These alleles have distinct patterns of RXLR effector recognition. While Rpi-chc1.1 recognized multiple PexRD12 (Avrchc1.1) proteins, Rpi-chc1.2 recognized multiple PexRD31 (Avrchc1.2) proteins, both belonging to the PexRD12/31 effector superfamily. Domain swaps between Rpi-chc1.1 and Rpi-chc1.2 revealed that overlapping subdomains in the leucine-rich repeat (LRR) domain are responsible for the difference in effector recognition. This study showed that Rpi-chc1.1 and Rpi-chc1.2 evolved to recognize distinct members of the same PexRD12/31 effector family via the LRR domain. The biased distribution of polymorphisms suggests that exchange of LRRs during host–pathogen co-evolution can lead to novel recognition specificities. These insights will guide future strategies to breed durable resistant varieties

Polyvalent Phage CoNShP-3 as a Natural Antimicrobial Agent Showing Lytic and Antibiofilm Activities against Antibiotic-Resistant Coagulase-Negative Staphylococci Strains

Synthetic antimicrobials have a negative impact on food quality and consumer health, which is why natural antimicrobials are urgently needed. Coagulase-negative staphylococci (CoNS) has gained considerable importance for food poisoning and infection in humans and animals, particularly in biofilms. As a result, this study was conducted to control the CoNS isolated from food samples in Egypt. CoNS isolates were selected on the basis of their antibiotic susceptibility profiles and their biofilm-associated behavior. In this context, a total of 29 different bacteriophages were isolated and, in particular, lytic phages (6 isolates) were selected. The host range and physiological parameters of the lytic phages have been studied. Electron microscopy images showed that lytic phages were members of the families Myoviridae (CoNShP-1, CoNShP-3, and CoNSeP-2 isolates) and Siphoviridae (CoNShP-2, CoNSsP-1, and CoNSeP-1 isolates). CoNShP-1, CoNShP-2, and CoNShP-3 were found to be virulent to Staphylococcus haemolyticus, CoNSsP-1 to Staphylococcus saprophyticus and CoNSeP-1 and CoNSeP-2 to Staphylococcus epidermidis. Interestingly, the CoNShP-3 exhibited a typical polyvalent behavior, where not only lysis CoNS, but also other genera include Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA), Bacillus cereus and Bacillus subtilis. In addition, CoNShP-3 phage showed high stability at different temperatures and pH levels. Indeed, CoNShP-3 phage showed an antibiofilm effect against Staphylococcus epidermidis CFS79 and Staphylococcus haemolyticus CFS43, respectively, while Staphylococcus saprophyticus CFS28 biofilm was completely removed. Finally, CoNShP-3 phage demonstrated a high preservative efficacy over short and long periods of storage against inoculated CoNS in chicken breast sections. In conclusion, this study highlights the control of CoNS pathogens using a polyvalent lytic phage as a natural antibacterial and antibiofilm agent from a food safety perspective

Silencing of the tomato phosphatidylinositol‐phospholipase C2 (SlPLC2) reduces plant susceptibility to Botrytis cinerea

The tomato [Solanum lycopersicum (Sl)] phosphatidylinositol‐phospholipase C (PI‐PLC) gene family is composed of six members, named SlPLC1 to SlPLC6, differentially regulated on pathogen attack. We have previously shown that the fungal elicitor xylanase induces a raise of SlPLC2 and SlPLC5 transcripts and that SlPLC2, but not SlPLC5, is required for xylanase‐induced expression of defense‐related genes. In this work we studied the role of SlPLC2 in the interaction between tomato and the necrotrophic fungus Botrytis cinerea. Inoculation of tomato leaves with B. cinerea increases SlPLC2 transcript levels. We knocked‐down the expression of SlPLC2 by virus‐induced gene silencing and plant defense responses were analyzed upon B. cinerea inoculation. SlPLC2 silenced plants developed smaller necrotic lesions concomitantly with less proliferation of the fungus. Silencing of SlPLC2 resulted as well in a reduced production of reactive oxygen species. Upon B. cinerea inoculation, transcript levels of the salicylic acid (SA)‐defense pathway marker gene SlPR1a were diminished in SlPLC2 silenced plants compared to non‐silenced infected plants, while transcripts of the jasmonic acid (JA)‐defense gene markers Proteinase Inhibitor I and II (SlPI‐I and SlPI‐II) were increased. This implies that SlPLC2 participates in plant susceptibility to B. cinerea.Instituto de Fisiología y Recursos Genéticos VegetalesFil: Gonorazky, Ana Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Guzzo, Maria Carla. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Fisiología y Recursos Genéticos Vegetales; ArgentinaFil: Abd El Haliem, Ahmed M. Wageningen University. Laboratory of Phytopathology; HolandaFil: Joosten, Matthieu H. A. J. Wageningen University. Laboratory of Phytopathology; HolandaFil: Laxalt, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentin