A Novel Member of the Trehalose Transporter Family Functions as an H+-Dependent Trehalose Transporter in the Reabsorption of Trehalose in Malpighian Tubules

In insects, Malpighian tubules are functionally analogous to mammalian kidneys in that they not only are essential to excrete waste molecules into the lumen but also are responsible for the reabsorption of indispensable molecules, such as sugars, from the lumen to the principal cells. Among sugars, the disaccharide trehalose is highly important to insects because it is the main hemolymph sugar to serve as a source of energy and carbon. The trehalose transporter TRET1 participates in the transfer of newly synthesized trehalose from the fat body across the cellular membrane into the hemolymph. Although transport proteins must play a pivotal role in the reabsorption of trehalose in Malpighian tubules, the molecular context underlying this process remains obscure. Previously, we identified a Tret1 homolog (Nlst8) that is expressed principally in the Malpighian tubules of the brown planthopper (BPH). Here, we used the Xenopus oocyte expression system to show that NlST8 exerts trehalose transport activity that is elevated under low pH conditions. These functional assays indicate that Nlst8 encodes a proton-dependent trehalose transporter (H-TRET1). To examine the involvement of Nlst8 in trehalose reabsorption, we analyzed the sugar composition of honeydew by using BPH with RNAi gene silencing. Trehalose was detected in the honeydew as waste excreted from Nlst8-dsRNA-injected BPH under hyperglycemic conditions. However, trehalose was not expelled from GFP-dsRNA-injected BPH even under hyperglycemic conditions. We conclude that NlST8 could participate in trehalose reabsorption driven by a H(+) gradient from the lumen to the principal cells of the Malpighian tubules

Sugar transporter genes of the brown planthopper, Nilaparvata lugens: a facilitated glucose/fructose transporter. Insect Biochem Mol Biol

a b s t r a c t The brown planthopper (BPH), Nilaparvata lugens, attacks rice plants and feeds on their phloem sap, which contains large amounts of sugars. The main sugar component of phloem sap is sucrose, a disaccharide composed of glucose and fructose. Sugars appear to be incorporated into the planthopper body by sugar transporters in the midgut. A total of 93 expressed sequence tags (ESTs) for putative sugar transporters were obtained from a BPH EST database, and 18 putative sugar transporter genes (Nlst1e18) were identified. The most abundantly expressed of these genes was Nlst1. This gene has previously been identified in the BPH as the glucose transporter gene NlHT1, which belongs to the major facilitator superfamily. Nlst1, 4, 6, 9, 12, 16, and 18 were highly expressed in the midgut, and Nlst2, 7, 8, 10, 15, 17, and 18 were highly expressed during the embryonic stages. Functional analyses were performed using Xenopus oocytes expressing NlST1 or 6. This showed that NlST6 is a facilitative glucose/fructose transporter that mediates sugar uptake from rice phloem sap in the BPH midgut in a manner similar to NlST1

Identification of a Ribonucleoprotein Intermediate of Tomato Mosaic Virus RNA Replication Complex Formation

The replication of eukaryotic positive-strand RNA virus genomes occurs in the membrane-bound RNA replication complexes. Previously, we found that the extract of evacuolated tobacco BY-2 protoplasts (BYL) is capable of supporting the translation and subsequent replication of the genomic RNAs of plant positive-strand RNA viruses, including Tomato mosaic virus (ToMV). Here, to dissect the process that precedes the formation of ToMV RNA replication complexes, we prepared membrane-depleted BYL (mdBYL), in which the membranes were removed by centrifugation. In mdBYL, ToMV RNA was translated to produce the 130-kDa and 180-kDa replication proteins, but the synthesis of any ToMV-related RNAs did not occur. When BYL membranes were added back to the ToMV RNA-translated mdBYL after the termination of translation with puromycin, ToMV RNA was replicated. Using a replication-competent ToMV derivative that encodes the FLAG-tagged 180-kDa replication protein, it was shown by affinity purification that a complex that contained the 130-kDa and 180-kDa proteins and ToMV genomic RNA was formed after translation in mdBYL. When the complex was mixed with BYL membranes, ToMV RNA was replicated, which suggests that this ribonucleoprotein complex is an intermediate of ToMV RNA replication complex formation. We have named this ribonucleoprotein complex the “pre-membrane-targeting complex.” Our data suggest that the formation of the pre-membrane-targeting complex is coupled with the translation of ToMV RNA, while posttranslationally added exogenous 180-kDa protein and replication templates can contribute to replication and can be replicated, respectively. Based on these results, we discuss the mechanisms of ToMV RNA replication complex formation

Quantitative and Qualitative Involvement of P3N-PIPO in Overcoming Recessive Resistance against Clover yellow vein virus in Pea Carrying cyv1

In pea carrying cyv1, a recessive gene for resistance to Clover yellow vein virus (ClYVV), ClYVV isolate Cl-no30 was restricted to the initially infected cells, whereas isolate 90-1 Br2 overcame this resistance. We mapped the region responsible for breaking of cyv1-mediated resistance by examining infection of cyv1 pea with chimeric viruses constructed from parts of Cl-no30 and 90-1 Br2. The breaking of resistance was attributed to the P3 cistron, which is known to produce two proteins: P3, from the main open reading frame (ORF), and P3N-PIPO, which has the N-terminal part of P3 fused to amino acids encoded by a small open reading frame (ORF) called PIPO in the +2 reading frame. We introduced point mutations that were synonymous with respect to the P3 protein but nonsynonymous with respect to the P3N-PIPO protein, and vice versa, into the chimeric viruses. Infection of plants with these mutant viruses revealed that both P3 and P3N-PIPO were involved in overcoming cyv1-mediated resistance. Moreover, P3N-PIPO quantitatively affected the virulence of Cl-no30 in cyv1 pea. Additional expression in trans of the P3N-PIPO derived from Cl-no30, using White clover mosaic virus as a vector, enabled Cl-no30 to move to systemic leaves in cyv1 pea. Susceptible pea plants infected with chimeric ClYVV possessing the P3 cistron of 90-1 Br2, and which were therefore virulent toward cyv1 pea, accumulated more P3N-PIPO than did those infected with Cl-no30, suggesting that the higher level of P3N-PIPO in infected cells contributed to the breaking of resistance by 90-1 Br2. This is the first report showing that P3N-PIPO is a virulence determinant in plants resistant to a potyvirus

Truncated yet functional viral protein produced via RNA polymerase slippage implies underestimated coding capacity of RNA viruses

RNA viruses use various strategies to condense their genetic information into small genomes. Potyviruses not only use the polyprotein strategy, but also embed an open reading frame, pipo, in the P3 cistron in the -1 reading frame. PIPO is expressed as a fusion protein with the N-terminal half of P3 (P3N-PIPO) via transcriptional slippage of viral RNA-dependent RNA polymerase (RdRp). We herein show that clover yellow vein virus (ClYVV) produces a previously unidentified factor, P3N-ALT, in the +1 reading frame via transcriptional slippage at a conserved G(1-2)A(6-7) motif, as is the case for P3N-PIPO. The translation of P3N-ALT terminates soon, and it is considered to be a C-terminal truncated form of P3. In planta experiments indicate that P3N-ALT functions in cell-to-cell movement along with P3N-PIPO. Hence, all three reading frames are used to produce functional proteins. Deep sequencing of ClYVV RNA from infected plants endorses the slippage by viral RdRp. Our findings unveil a virus strategy that optimizes the coding capacity

Transfection of Protoplasts Prepared from Arabidopsis thaliana Leaves for Plant Virus Research

Antiviral Resistance in Plant