SummaryRoot-knot nematodes (RKNs) induce giant cells (GCs) from root vascular cells inside the galls. Accompanying molecular changes as a function of infection time and across different species, and their functional impact, are still poorly understood. Thus, the transcriptomes of tomato galls and laser capture microdissected (LCM) GCs over the course of parasitism were compared with those of Arabidopsis, and functional analysis of a repressed gene was performed.Microarray hybridization with RNA from galls and LCM GCs, infection-reproduction tests and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) transcriptional profiles in susceptible and resistant (Mi-1) lines were performed in tomato.Tomato GC-induced genes include some possibly contributing to the epigenetic control of GC identity. GC-repressed genes are conserved between tomato and Arabidopsis, notably those involved in lignin deposition. However, genes related to the regulation of gene expression diverge, suggesting that diverse transcriptional regulators mediate common responses leading to GC formation in different plant species. TPX1, a cell wall peroxidase specifically involved in lignification, was strongly repressed in GCs/galls, but induced in a nearly isogenic Mi-1 resistant line on nematode infection. TPX1 overexpression in susceptible plants hindered nematode reproduction and GC expansion.Time-course and cross-species comparisons of gall and GC transcriptomes provide novel insights pointing to the relevance of gene repression during RKN establishment.

Root-knot nematodes feed from specialized giant cells induced in the plants that they parasitize. We found that the promoter of the Hahsp17.7G4 gene, which encodes a small heat-shock protein involved in embryogenesis and stress responses, directed GUS expression in tobacco galls induced by the root-knot nematode Meloidogyne incognita. In roots containing a GUS reporter fusion to the Hahsp17.7G4 promoter, 10% of the galls stained for GUS expression 1 to 3 days after infection and the fraction stained increased to 60 to 80% 17 to 20 days after infection. A DNA fragment from -83 to +163, which contains heat-shock element (HSE) core sequences, is sufficient to support a promoter activity largely restricted to giant cells within the galls. Two-point mutations in HSE cores, previously reported to abolish the heat-shock response and to strongly reduce the embryogenesis response of the same promoter, did not reduce expression in giant cells. This suggests a distinct regulation of the promoter by nematodes. However, additional point mutations located at positions crucial for binding of heat-shock transcription factors (HSFs) caused a severe decrease in the nematode response. These results demonstrate that HSEs are involved in the promoter activation in giant cells and suggest that HSFs may mediate this response.

Plant organ gene expression profile analyses are complicated by the various cell types, and therefore transcription patterns, present in each organ. For example, each gall formed in roots following root knot nematode infection contains between four and eight specialized feeding cells (giant cells, GCs) embedded within hypertrophied root tissues. A recent goal in plant science has been the isolation of nematode feeding cell mRNAs for subsequent gene expression analysis. By adapting current protocols for different plant species and cells, we have developed a simple and rapid method for obtaining GCs from frozen tissue sections of tomato with good morphology and preserved RNA. The tissue sections obtained were suitable for the laser capture microdissection of GCs 6-7 days post-infection, and even of very early developing GCs (48-72 h post-infection), by fixation of tissue with ethanol-acetic acid, infiltration with sucrose and freezing in isopentane with optimal cutting temperature medium. This process was also successful for obtaining control vascular cells from uninfected roots for direct comparison with GCs. A minimum of about 300 GCs and 600 control vascular cells was required for efficient linear RNA amplification through in vitro transcription. Laser capture microdissection-derived RNA, after two rounds of amplification, was successfully used for microarray hybridization and validated with several differentially expressed genes by quantitative polymerase chain reaction. Consistent with our results, 117 homologous genes were found to be co-regulated in a previous microarray analysis of Arabidopsis galls at the same developmental stage. Therefore, we conclude that our method allows the isolation of a sufficient quantity of RNA with a high quality/integrity, appropriate for differential transcriptome analysis.

Highly sensitive techniques for transcriptome analysis, such as microarrays, complementary DNA-amplified fragment length polymorphisms (cDNAAFLPs), and others currently used in functional genomics require a high RNA quality and integrity, as well as reproducibility among extractions of replicates from the same tissue. There are, however, few technical papers comparing different homogenization techniques and reagents to extract RNA from small quantities of plant tissue. We extracted RNA from tomato seedlings with the three different commercial reagents TRIZOL LS Ò , TRIZOL Ò , and TRI Reagent Ò in combination with pulverization, homogenizationmaceration in a mortar, and homogenization with mild vibration plus glass beads, and evaluated total RNA integrity-quality and yield. Pulverization under liquid nitrogen combined with TRIZOL LS Ò as extraction reagent and homogenization-maceration in mortar with TRI Reagent Ò , are the procedures that rendered higher RNA yield, integrity and quality, as well as reproducibility among independent RNA extractions. In contrast, short mild vibration pulses (4500 r.p.m. for 5 s) mixed with glass beads, rendered low extraction efficiency and caused, in most cases, partial RNA degradation.

Obligate sedentary endoparasitic nematodes, such as the root-knot and cyst nematodes, elicit the differentiation of specialized nematode nurse or feeding cells [nematode feeding sites (NFS), giant cells and syncytia, respectively]. During NFS differentiation, marked changes in cell cycle progression occur, partly similar to those induced by some geminiviruses. In this work, we describe the activation of V-sense promoters from the Maize streak virus (MSV) and Wheat dwarf virus (WDV) in NFS formed by root-knot and cyst nematodes. Both promoters were transiently active in microinjection experiments. In tobacco and Arabidopsis transgenic lines carrying promoter-beta-glucuronidase fusions, the MSV V-sense promoter was activated in the vascular tissues of aerial plant parts, primarily leaf and cotyledon phloem tissue and some floral structures. Interestingly, in roots, promoter activation was restricted to syncytia and giant cells tested with four different nematode populations, but undetectable in the rest of the root system. As the activity of the promoter in transgenic rootstocks should be restricted to NFS only, the MSV promoter may have utility in engineering grafted crops for nematode control. Therefore, this study represents a step in the provision of some of the much needed additional data on promoters with restricted activation in NFS useful in biotechnological nematode control strategies.