ABSTRACT: Root-knot nematodes (RKN; Meloidogyne spp.) are sedentary parasites that affect a high variety of plants with a negative impact in the production of crops such as tomato. The infective RKN induces in the roots 4-7 highly specialized feeding cells (giant cells, GCs) developed into a hypertrophy cellular tissue or visible root swelling called M-bM-^@M-^\gallM-bM-^@M-^]. During GCs differentiation drastic alterations in genes expression occurs. However, information on genome-wide transcriptional profiles specifically in GCs is still lacking. With the aim to identify potential targets specifically regulated in GCs, we performed a temporal and spatial differential transcription profiling of tomato (Solanum lycopersicum), during the course of Meloidogyne javanica infection hybridizing TOM1 microarrays with hand-dissected galls at 1, 3, 7 and 14 days post-infection (dpi) and GCs exclusively isolated by LASER CAPTURE MICRODISSECTION (LCM) at 3 and 7 dpi. A GCs and galls comparison, at the same stage of development, reveals clear differences between their transcriptional patterns. For this purpose, a fast and efficient method to isolate LCM GCs from cryopreserved galls in the earliest differentiation state described to date: 48-72-hour post-infection (3 dpi), allowing good RNA preservation, was developed. In galls transcriptomic anaylsis, about 3000 galls and 4400 control uninfected root fragments were carefully hand-dissected at 1, 3, 7 and 14 dpi. Thus, more than 12 plates with 8 individual infected plants each, and 18 plates with the same number of plants as controls were used for only one independent experiment in each time point. Total RNA from two independent experiments was equimolarly pooled at each time of infection, in order to achieve a biological replicate for galls or control regardless. Therefore, a total of 6 independent experiments were performed to obtain 3 independent biological replicates for each infection point of galls and control analyzed. Following isolation, total RNA pooled from galls and control were subjected to a single round of linear amplification. In GCs transcriptomic analysis, independent biological replicates of aRNA from 300 GCs versus 600 control cells (CCs) from control root LCM at 3 and 7 dpi were obtained. Fifty GCs and 100 CCs were independently captured from sections onto separate CapSure HS LCM caps, and total RNA isolation was performed from each cap. RNA from six separate caps was pooled for a single biological replicate of GCs or CCs, respectively. In total, three independent biological replicates were processed. Thus, near to 7000 individual cells were harvested by LCM. The material was obtained from more that 18 independent infection experiments.
