The effect of cucumber mosaic virus (CMV) infection on the phloem sap mRNA population was investigated in pumpkin Cucurbita maxima Duch. cv. Big Max, through analysis of a suppressive subtractive hybridisation (SSH) library. Analysis of the infected phloem library identified 91 highly diverse mRNA species, including enzymes involved in general metabolism, transcription factors and signalling agents. Our analysis indicated that, quantitatively, the effect of CMV infection on the composition of the phloem sap transcriptome was minor in nature. Virtual northern analysis was used to confirm the specific upregulation of these transcripts in the phloem of CMV-infected plants. In silico northern analysis also confirmed that none of the transcripts identified in the SSH library was contained in the population of mRNA species present in the phloem sap of healthy plants. Induction levels ranged from low to high and in situ hybridisation studies showed that transcripts displayed a range of accumulation patterns. Collectively, our findings suggest that plants have evolved a highly robust mechanism for the exchange of information macromolecules between the companion cell (CC) and the sieve tube system. Production of viral movement protein (MP) in the CC is not sufficient for the indiscriminate transport of mRNA into the sieve element. Our findings are discussed in the context of symptom development and likely strong selection pressure, on the viral genome, to encode for a MP that does not adversely interfere with the phloem long-distance trafficking system.
Quantitation of mRNA species by RT-PCR on total mRNA population.
