PHLOEM PROTEOMICS IN STUDYING HOST VIRUS INTERACTION C.Anuradha and R.

Selvarajan Molecular Virology Lab, National Research Centre for Banana, Trichy, Tamil Nadu

Phloem tissue is traditionally considered responsible for the translocation of photoassimilates from source to distant sink organs. In angiosperms, the phloem is a complex tissue, composed of several structurally and functionally different cell types, which is almost always associated with the xylem. After the unequal division of a phloem mother cell, the two cells undergo unique differentiation to form the companion cell sieve element (CC-SEs) complex. Differentiation of the SE is characterized by loss of organelles, including nuclei and ribosomes, changes in the formation of the endoplasmic reticulum (ER), and widening of the symplasmic connections between SEs to form the sieve place pores. Sieve tubes are transport conduits not only for photo assimilates but also for macromolecules and other compounds that are involved in sieve tube maintenance and systemic signaling. Functional analysis of proteins and transcripts identified in phloem exudates revealed a wide range of processes including metabolism, responses to stress, transport, detoxification of reactive oxygen species (ROS), DNA/RNA binding, signaling and protein turnover. Recent studies have also revealed the presence of small RNA molecules, including microRNAs (miRNAs), in phloem exudates from cucurbits, Brassica napus and Malus domestica. Present proteomics research aims both at identifying new proteins in relation to their function and ultimately at unravelling how their expression is controlled with in regulatory networks. The expression or function of proteins is modulated at many points from transcription to post-translation, which generally cannot be predicted from analysis of nucleic acids alone. There is poor correlation between the abundance of mRNA transcribed from the DNA, the respective proteins translated from that mRNA and the transcript can be spliced in various ways to yield different protein forms. Extensive changes can also be introduced during or after translation for example, the addition of specific carbohydrate side chains or phosphorylation leading to multiple protein products from a single gene. Systematic analysis of all protein expression patterns and protein sequences in different tissues, cells and subcellular fractions, will play an important role in studying many different aspects of plant function. Much of the regulation of physiological processes occur post-transcriptionally, the

measurement of protein expression by post-genomic approaches is essential to give a more accurate and comprehensive picture of cellular activity. Virus replication takes place in the nucleus containing phloem parenchyma and companion cells that surround the enucleate sieve elements (SE). Long distance movement of virions occurs via the sieve tubes formed by connecting SE. Virus particles have been observed in the branched plasmodesmata connecting companion cells and SE suggesting, that virions are the infectious entity moving over long distances. Systemic spread of plant viruses also involves long distance trafficking of the virus pariticles with in the sieve tube. Most often, the viral material is trafficked long distances as their ribonucleoprotein complexes or virions consisting of an outer protein shell (capsid) and an inner nucleic acid core. Similar to other sieve tube macromolecules, virus particles are transported from the CCs into the SEs via the special plasmodesmata (PD) interconnecting these cell types. As no evidence exists for viral exit to the apoplast, and ultrastructural studies indicate the existence of PD between phloem parenchyma and CCs in all studied species, it is accepted that viruses enter the sieve tube symplastically. Several identified phloem sap proteins have been characterized as RNA-binding proteins. In vitro interaction was demonstrated between cucumber PP2, a dimeric lectin and Hop stunt viroid. A melon phloem lectin was found to interact with viral RNA, as well as with its own mRNA. Interaction between a phloem protein and its mRNA was also demonstrated for the pumpkin cmPP16. CmPP16 was shown to mediate selective transport of its mRNA through PD. These findings suggest that specific phloem proteins are involved in the trafficking of RNA molecules from the CC to the sieve tube for their long-distance translocation. Phloem proteins can bind to pathogens, such as phytoviruses or viriods. The role of viral movement protein (MP) in the cell-to-cell trafficking of viruses is well established. Expression of the tobacco mosaic virus MP in transgenic tobacco plants causes a significant increase in the size-exclusion limit (SEL) of PD interconnecting these cells. The phloem protein 1-homolog from Cucumis melo was shown to bind to Cucumber Mosaic Virus (CMV) particles invivo and to increase virus stability. Two predominant lectins in cucumber sap, belonging to the phloem-protein 2 family (PP2), were also shown to bind to viroids and virus RNA in vitro and in vivo, a property that was proposed to facilitate long-distance movement of these entities. Because the SE is not only used by luteo virids to move over long distances but also contains the virus stock for phloem feeding insects, for reasoned that phloem

proteins might not only have an effect on virus movement in planta but also could play a role in virus transmission by aphids. Virus particle transmission from plant to plant, are taken up exclusively from SE by the phloem-feeding aphids during sap ingestion. Implication of plant proteins in the aphid transmission process is so far limited to the non-persistently transmitted cauliflower mosaic virus (CaMV). Several proteins from cucumber SE can interact with cucurbit aphid borne yellows virus (CABYV). Phloem lectins can stimulate virus transmission by aphid when mixed with virus in an artificial diet and have a protective activity of the virions against in vitro degradation. However, this effect on virus transmission rate was not restricted to plant lectins and could be extended to various non-plant proteins, suggesting that any protein present in phloem sap in sufficiently high concentration could stimulate virus transmission by aphid. During the infection process, the presence of the viral MP within the CC could, in principle, result in the aberrant long-distance transport of cell-autonomous RNA molecules. Such events could have an impact on normal plant growth and development. Indeed, changes to the population of long distance mRNA may well contribute to viral-induced symptom development. Therefore, studying of phloem proteins is very much important in understanding the host pathogen interaction.