Identification of Tomato microRNAs in Late Response to Trichoderma atroviride.

The tomato (Solanum lycopersicum) is an important crop worldwide and is considered a model plant to study stress responses. Small RNAs (sRNAs), 21-24 nucleotides in length, are recognized as a conserved mechanism for regulating gene expression in eukaryotes. Plant endogenous sRNAs, such as microRNA (miRNA), have been involved in disease resistance. High-throughput RNA sequencing was used to analyze the miRNA profile of the aerial part of 30-day-old tomato plants after the application of the fungus Trichoderma atroviride to the seeds at the transcriptional memory state. Compared to control plants, ten differentially expressed (DE) miRNAs were identified in those inoculated with Trichoderma, five upregulated and five downregulated, of which seven were known (miR166a, miR398-3p, miR408, miR5300, miR6024, miR6027-5p, and miR9471b-3p), and three were putatively novel (novel miR257, novel miR275, and novel miR1767). miRNA expression levels were assessed using real-time quantitative PCR analysis. A plant sRNA target analysis of the DE miRNAs predicted 945 potential target genes, most of them being downregulated (84%). The analysis of KEGG metabolic pathways showed that most of the targets harbored functions associated with plant-pathogen interaction, membrane trafficking, and protein kinases. Expression changes of tomato miRNAs caused by Trichoderma are linked to plant defense responses and appear to have long-lasting effects.

Food systems microbiome-related educational needs.

Within the European-funded Coordination and Support Action MicrobiomeSupport (https://www.microbiomesupport.eu/), the Workshop 'Education in Food Systems Microbiome Related Sciences: Needs for Universities, Industry and Public Health Systems' brought together over 70 researchers, public health and industry partners from all over the world to work on elaborating microbiome-related educational needs in food systems. This publication provides a summary of discussions held during and after the workshop and the resulting recommendations.

Divergent regulation of auxin responsive genes in root-knot and cyst nematodes feeding sites formed in Arabidopsis.

Cysts (CNs) and root-knot nematodes (RKNs) induce specialized feeding cells, syncytia, and giant cells (GCs), respectively, within plant roots. The plant tissues around the GCs usually by respond forming a root swelling called a gall that contains the GCs. The ontogenesis of feeding cells is different. GC formation is a process of new organogenesis from vascular cells, which are still not well characterized, that differentiate into GCs. In contrast, syncytia formation involves the fusion of adjacent cells that have already differentiated. Nonetheless, both feeding sites show an auxin maximum pertinent to feeding site formation. However, data on the molecular divergences and similarities between the formation of both feeding sites regarding auxin-responsive genes are still scarce. We studied genes from the auxin transduction pathways that are crucial during gall and lateral root (LR) development in the CN interaction by using promoter-reporter (GUS/LUC)transgenic lines, as well as loss of function lines of Arabidopsis. The promoters pGATA23 and several deletions of pmiR390a were active in syncytia, as were in galls, but pAHP6 or putative up-stream regulators as ARF5/7/19 were not active in syncytia. Additionally, none of these genes seemed to play a key role during cyst nematode establishment in Arabidopsis, as the infection rates in loss of function lines did not show significant differences compared to control Col-0 plants. Furthermore, the presence of only canonical AuxRe elements in their proximal promoter regions is highly correlated with their activation in galls/GCs (AHP6, LBD16), but those promoters active in syncytia (miR390, GATA23) carry AuxRe overlapping core cis-elements for other transcription factor families (i.e., bHLH, bZIP). Strikingly, in silico transcriptomic analysis showed very few genes upregulated by auxins common to those induced in GCs and syncytia, despite the high number of upregulated IAA responsive genes in syncytia and galls. The complex regulation of auxin transduction pathways, where different members of the auxin response factor (ARF) family may interact with other factors, and the differences in auxin sensitivity, as indicated by the lower induction of the DR5 sensor in syncytia than galls, among other factors, may explain the divergent regulation of auxin responsive genes in the two types of nematode feeding sites.

Microbiome Research as an Effective Driver of Success Stories in Agrifood Systems - A Selection of Case Studies.

Increasing knowledge of the microbiome has led to significant advancements in the agrifood system. Case studies based on microbiome applications have been reported worldwide and, in this review, we have selected 14 success stories that showcase the importance of microbiome research in advancing the agrifood system. The selected case studies describe products, methodologies, applications, tools, and processes that created an economic and societal impact. Additionally, they cover a broad range of fields within the agrifood chain: the management of diseases and putative pathogens; the use of microorganism as soil fertilizers and plant strengtheners; the investigation of the microbial dynamics occurring during food fermentation; the presence of microorganisms and/or genes associated with hazards for animal and human health (e.g., mycotoxins, spoilage agents, or pathogens) in feeds, foods, and their processing environments; applications to improve HACCP systems; and the identification of novel probiotics and prebiotics to improve the animal gut microbiome or to prevent chronic non-communicable diseases in humans (e.g., obesity complications). The microbiomes of soil, plants, and animals are pivotal for ensuring human and environmental health and this review highlights the impact that microbiome applications have with this regard.

Root-knot nematodes induce gall formation by recruiting developmental pathways of post-embryonic organogenesis and regeneration to promote transient pluripotency.

Root-knot nematodes (RKNs; Meloidogyne spp.) induce new post-embryogenic organs within the roots (galls) where they stablish and differentiate nematode feeding cells, giant cells (GCs). The developmental programmes and functional genes involved remain poorly defined. Arabidopsis root apical meristem (RAM), lateral root (LR) and callus marker lines, SHORT-ROOT/SHR, SCARECROW/SCR, SCHIZORIZA/SCZ, WUSCHEL-RELATED-HOMEOBOX-5/WOX5, AUXIN-RESPONSIVE-FACTOR-5/ARF5, ARABIDOPSIS-HISTIDINE PHOSPHOTRANSFER-PROTEIN-6/AHP6, GATA-TRANSCRIPTION FACTOR-23/GATA23 and S-PHASE-KINASE-ASSOCIATED-PROTEIN2B/SKP2B, were analysed for nematode-dependent expression. Their corresponding loss-of-function lines, including those for LR upstream regulators, SOLITARY ROOT/SLR/IAA14, BONDELOS/BDL/IAA12 and INDOLE-3-ACETIC-ACID-INDUCIBLE-28/IAA28, were tested for RKN resistance/tolerance. LR genes, for example ARF5 (key factor for root stem-cell niche regeneration), GATA23 (which specifies pluripotent founder cells) and AHP6 (cytokinin-signalling-inhibitor regulating pericycle cell-divisions orientation), show a crucial function during gall formation. RKNs do not compromise the number of founder cells or LR primordia but locally induce gall formation possibly by tuning the auxin/cytokinin balance in which AHP6 might be necessary. Key RAM marker genes were induced and functional in galls. Therefore, the activation of plant developmental programmes promoting transient-pluripotency/stemness leads to the generation of quiescent-centre and meristematic-like cell identities within the vascular cylinder of galls. Nematodes enlist developmental pathways of new organogenesis and/or root regeneration in the vascular cells of galls. This should determine meristematic cell identities with sufficient transient pluripotency for gall organogenesis.

A role for ALF4 during gall and giant cell development in the biotic interaction between Arabidopsis and Meloidogyne spp.

Root-knot nematodes (RKNs; Meloidogyne spp.) are a major pest for the agriculture worldwide. RKNs induce specialized feeding cells (giant cells, GCs) inside galls which are de novo formed pseudo-organs in the roots that share similarities with other developmental processes as lateral root (LR) and callus formation or grafting involving new vascular development or pericycle proliferation. Hence, it is pertinent to study the molecular mechanisms directing the plant-nematode interaction. In this respect, ALF4 is a key gene during LR formation, vascular vessels reconnection in grafting, hormone-induced callus formation or de novo root organogenesis from leaf explants. Our results show that ALF4 is also induced in galls at early infection stages in an auxin-independent way. Furthermore, ALF4 activity is necessary for the formation of proper galls and GCs, as the mutant alf4-1 presents aberrant galls and GCs with severe structural abnormalities leading to a dramatic reduction in the nematode egg production. However, a low-reproduction rate is maintained, that might be explained by the local auxin maximum build by the nematodes in galls, partially rescuing alf4-1 phenotype. This would be similar to the partial rescue described for LR formation with exogenous auxins and also agrees with the LR emergence from alf4-1 galls but not from uninfected roots. In addition, ALF4 is also induced in syncytia formed by cyst nematodes. All these data support a pivotal role for ALF4 during de novo organogenesis processes induced by endoparasitic nematodes, in addition to its role in LR formation, callus development or vessel reconnection during grafting.

A Phenotyping Method of Giant Cells from Root-Knot Nematode Feeding Sites by Confocal Microscopy Highlights a Role for CHITINASE-LIKE 1 in Arabidopsis.

Most effective nematicides for the control of root-knot nematodes are banned, which demands a better understanding of the plant-nematode interaction. Understanding how gene expression in the nematode-feeding sites relates to morphological features may assist a better characterization of the interaction. However, nematode-induced galls resulting from cell-proliferation and hypertrophy hinders such observation, which would require tissue sectioning or clearing. We demonstrate that a method based on the green auto-fluorescence produced by glutaraldehyde and the tissue-clearing properties of benzyl-alcohol/benzyl-benzoate preserves the structure of the nematode-feeding sites and the plant-nematode interface with unprecedented resolution quality. This allowed us to obtain detailed measurements of the giant cells' area in an Arabidopsis line overexpressing CHITINASE-LIKE-1 (CTL1) from optical sections by confocal microscopy, assigning a role for CTL1 and adding essential data to the scarce information of the role of gene repression in giant cells. Furthermore, subcellular structures and features of the nematodes body and tissues from thick organs formed after different biotic interactions, i.e., galls, syncytia, and nodules, were clearly distinguished without embedding or sectioning in different plant species (Arabidopsis, cucumber or Medicago). The combination of this method with molecular studies will be valuable for a better understanding of the plant-biotic interactions.

Root-Knot and Cyst Nematodes Activate Procambium-Associated Genes in Arabidopsis Roots.

Developmental plasticity is one of the most striking features of plant morphogenesis, as plants are able to vary their shapes in response to environmental cues. Biotic or abiotic stimuli often promote organogenesis events in plants not observed under normal growth conditions. Root-knot nematodes (RKNs) are known to parasitize multiple species of rooting plants and to induce characteristic tissue expansion called galls or root-knots on the roots of their hosts by perturbing the plant cellular machinery. Galls contain giant cells (GCs) and neighboring cells, and the GCs are a source of nutrients for the parasitizing nematode. Highly active cell proliferation was observed in galls. However, the underlying mechanisms that regulate the symptoms triggered by the plant-nematode interaction have not yet been elucidated. In this study, we deciphered the molecular mechanism of gall formation with an in vitro infection assay system using RKN Meloidogyne incognita, and the model plant Arabidopsis thaliana. By taking advantages of this system, we performed next-generation sequencing-based transcriptome profiling, and found that the expression of procambium identity-associated genes were enriched during gall formation. Clustering analyses with artificial xylogenic systems, together with the results of expression analyses of the candidate genes, showed a significant correlation between the induction of gall cells and procambium-associated cells. Furthermore, the promoters of several procambial marker genes such as ATHB8, TDR and WOX4 were activated not only in M. incognita-induced galls, but similarly in M. javanica induced-galls and Heterodera schachtii-induced syncytia. Our findings suggest that phytoparasitic nematodes modulate the host's developmental regulation of the vascular stem cells during gall formation.

Molecular Transducers from Roots Are Triggered in Arabidopsis Leaves by Root-Knot Nematodes for Successful Feeding Site Formation: A Conserved Post-Embryogenic De novo Organogenesis Program?

Root-knot nematodes (RKNs; Meloidogyne spp.) induce feeding cells (giant cells; GCs) inside a pseudo-organ (gall) from still unknown root cells. Understanding GCs ontogeny is essential to the basic knowledge of RKN-plant interaction and to discover novel and effective control strategies. Hence, we report for the first time in a model plant, Arabidopsis, molecular, and cellular features concerning ectopic de novo organogenesis of RKNs GCs in leaves. RKNs induce GCs in leaves with irregular shape, a reticulated cytosol, and fragmented vacuoles as GCs from roots. Leaf cells around the nematode enter G2-M shown by ProCycB1;1:CycB1;1(NT)-GUS expression, consistent to multinucleated GCs. In addition, GCs nuclei present irregular and varied sizes. All these characteristics mentioned, being equivalent to GCs in root-galls. RKNs complete their life cycle forming a gall/callus-like structure in the leaf vascular tissues resembling auxin-induced callus with an auxin-response maxima, indicated by high expression of DR5::GUS that is dependent on leaf auxin-transport. Notably, induction of leaves calli/GCs requires molecular components from roots crucial for lateral roots (LRs), auxin-induced callus and root-gall formation, i.e., LBD16. Hence, LBD16 is a xylem pole pericycle specific and local marker in LR primordia unexpectedly induced locally in the vascular tissue of leaves after RKN infection. LBD16 is also fundamental for feeding site formation as RKNs could not stablish in 35S::LBD16-SRDX leaves, and likely it is also a conserved molecular hub between biotic and developmental signals in Arabidopsis either in roots or leaves. Moreover, RKNs induce the ectopic development of roots from leaf and root-galls, also formed in mutants compromised in LR formation, arf7/arf19, slr, and alf4. Therefore, nematodes must target molecular signatures to induce post-embryogenic de novo organogenesis through the LBD16 callus formation pathway partially different from those prevalent during normal LR development.

A Standardized Method to Assess Infection Rates of Root-Knot and Cyst Nematodes in Arabidopsis thaliana Mutants with Alterations in Root Development Related to Auxin and Cytokinin Signaling.

Plant parasitic nematodes cause a great impact in agricultural systems. The search for effective control methods is partly based on the understanding of underlying molecular mechanisms leading to the formation of nematode feeding sites. In this respect, crosstalk of hormones such as auxins and cytokinins (IAA, CK) between the plant and the nematode seems to be crucial. Thence, the study of loss of function or overexpressing lines with altered IAA and CK functioning is entailed. Those lines frequently show developmental defects in the number, position and/or length of the lateral roots what could generate a bias in the interpretation of the nematode infection parameters. Here we present a protocol to assess differences in nematode infectivity with the lowest interference of root architecture phenotypes in the results. Thus, tailored growth conditions and normalization parameters facilitate the standardized phenotyping of nematode infection.

Long-Term In Vitro System for Maintenance and Amplification of Root-Knot Nematodes in Cucumis sativus Roots.

Root-knot nematodes (RKN) are polyphagous plant-parasitic roundworms that produce large crop losses, representing a relevant agricultural pest worldwide. After infection, they induce swollen root structures called galls containing giant cells (GCs) indispensable for nematode development. Among efficient control methods are biotechnology-based strategies that require a deep knowledge of underlying molecular processes during the plant-nematode interaction. Methods of achieving this knowledge include the application of molecular biology techniques such as transcriptomics (as massive sequencing or microarray hybridization), proteomics or metabolomics. These require aseptic experimental conditions, as undetected contamination with other microorganisms could compromise the interpretation of the results. Herein, we present a simple, efficient and long-term method for nematode amplification on cucumber roots grown in vitro. Amplification of juveniles (J2) from the starting inoculum is around 40-fold. The method was validated for three Meloidogyne species (Meloidogyne javanica, M. incognita, and M. arenaria), producing viable and robust freshly hatched J2s. These J2s can be used for further in vitro infection of different plant species such as Arabidopsis, tobacco and tomato, as well as to maintain and amplify the population. The method allowed maintenance of around 90 Meloidogyne sp. generations (one every 2 months) from a single initial female over 15 years.