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 the gene regulatory module microRNA172/TARGET OF EARLY ACTIVATION TAGGED 1/FLOWERING LOCUS T (miRNA172/TOE1/FT) in the feeding sites induced by Meloidogyne javanica in Arabidopsis thaliana.

Root knot nematodes (RKNs) penetrate into the root vascular cylinder, triggering morphogenetic changes to induce galls, de novo formed 'pseudo-organs' containing several giant cells (GCs). Distinctive gene repression events observed in early gall/GCs development are thought to be mediated by post-transcriptional silencing via microRNAs (miRNAs), a process that is far from being fully characterized. Arabidopsis thaliana backgrounds with altered activities based on target 35S::MIMICRY172 (MIM172), 35S::TARGET OF EARLY ACTIVATION TAGGED 1 (TOE1)-miR172-resistant (35S::TOE1R ) and mutant (flowering locus T-10 (ft-10)) lines were used for functional analysis of nematode infective and reproductive parameters. The GUS-reporter lines, MIR172A-E::GUS, treated with auxin (IAA) and an auxin-inhibitor (a-(phenyl ethyl-2-one)-indole-3-acetic acid (PEO-IAA)), together with the MIR172C AuxRE::GUS line with two mutated auxin responsive elements (AuxREs), were assayed for nematode-dependent gene expression. Arabidopsis thaliana backgrounds with altered expression of miRNA172, TOE1 or FT showed lower susceptibility to the RKNs and smaller galls and GCs. MIR172C-D::GUS showed restricted promoter activity in galls/GCs that was regulated by auxins through auxin-responsive factors. IAA induced their activity in galls while PEO-IAA treatment and mutations in AuxRe motifs abolished it. The results showed that the regulatory module miRNA172/TOE1/FT plays an important role in correct GCs and gall development, where miRNA172 is modulated by auxins.

Phenotyping nematode feeding sites: three-dimensional reconstruction and volumetric measurements of giant cells induced by root-knot nematodes in Arabidopsis.

The control of plant parasitic nematodes is an increasing problem. A key process during the infection is the induction of specialized nourishing cells, called giant cells (GCs), in roots. Understanding the function of genes required for GC development is crucial to identify targets for new control strategies. We propose a standardized method for GC phenotyping in different plant genotypes, like those with modified genes essential for GC development. The method combines images obtained by bright-field microscopy from the complete serial sectioning of galls with TrakEM2, specialized three-dimensional (3D) reconstruction software for biological structures. The volumes and shapes from 162 3D models of individual GCs induced by Meloidogyne javanica in Arabidopsis were analyzed for the first time along their life cycle. A high correlation between the combined volume of all GCs within a gall and the total area occupied by all the GCs in the section/s where they show maximum expansion, and a proof of concept from two Arabidopsis transgenic lines (J0121 â‰« DTA and J0121 â‰« GFP) demonstrate the reliability of the method. We phenotyped GCs and developed a reliable simplified method based on a two-dimensional (2D) parameter for comparison of GCs from different Arabidopsis genotypes, which is also applicable to galls from different plant species and in different growing conditions, as thickness/transparency is not a restriction.

A role for LATERAL ORGAN BOUNDARIES-DOMAIN 16 during the interaction Arabidopsis-Meloidogyne spp. provides a molecular link between lateral root and root-knot nematode feeding site development.

Plant endoparasitic nematodes induce the formation of their feeding cells by injecting effectors from the esophageal glands into root cells. Although vascular cylinder cells seem to be involved in the formation of root-knot nematode (RKN) feeding structures, molecular evidence is scarce. We address the role during gall development of LATERAL ORGAN BOUNDARIES-DOMAIN 16 (LBD16), a key component of the auxin pathway leading to the divisions in the xylem pole pericycle (XPP) for lateral root (LR) formation. Arabidopsis T-DNA tagged J0192 and J0121 XPP marker lines, LBD16 and DR5::GUS promoter lines, and isolated J0192 protoplasts were assayed for nematode-dependent gene expression. Infection tests in LBD16 knock-out lines were used for functional analysis. J0192 and J0121 lines were activated in early developing galls and giant cells (GCs), resembling the pattern of the G2/M-transition specific ProC yc B 1;1 :CycB1;1(NT)-GUS line. LBD16 was regulated by auxins in galls as in LRs, and induced by RKN secretions. LBD16 loss of function mutants and a transgenic line with defective XPP cells showed a significantly reduced infection rate. The results show that genes expressed in the dividing XPP, particularly LBD16, are important for gall formation, as they are for 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.

A Reliable Protocol for In situ microRNAs Detection in Feeding Sites Induced by Root-Knot Nematodes.

Galls induced by Meloidogyne spp. in plant roots are a complex organ formed by heterogeneous tissues; within them there are 5-8 giant cells (GCs) that root-knot nematodes use for their own nurturing. Subtle regulatory mechanisms likely mediate the massive gene repression described at early infection stages in galls, particularly in giant cells. Some of these mechanisms are mediated by microRNAs (miRNAs); hence we describe a reliable protocol to detect miRNAs abundance within the gall tissues induced by Meloidogyne spp. Some methods are available to determine the abundance of specific miRNAs in different plant parts; however, galls are complex organs formed by different tissues. Therefore, detection of miRNAs at the cellular level is particularly important to understand specific regulatory mechanisms operating within the GCs. In situ hybridization (ISH) is a classical, robust and accurate method that allows the localization of specific RNAs directly on plant tissues. We present for the first time an adapted and standardized ISH protocol to detect miRNAs in GCs induced by nematodes based on tissue embedded in paraffin and on-slide ISH of miRNAs. It can be adapted to any laboratory with no more requirements than a microtome and an optical microscope and it takes 10 days to perform once plant material has been collected. It showed to be very valuable for a quick detection of miRNAs expression pattern in tomato. We tested the protocol for miR390, as massive sequencing analysis showed that miR390 was induced at 3 dpi (days post-infection) in Arabidopsis galls and miR390 is 100% conserved between Arabidopsis and tomato. Successful localization of miR390 in tomato GCs constitutes a validation of this method that could be easily extended to other crops and/or syncytia induced by cyst nematodes. Finally, the protocol also includes guidance on troubleshooting.

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.