Plant cells contain proteins that are members of the major intrinsic protein (MIP) family, an ancient family of membrane channel proteins characterized by six membrane-spanning domains and two asparagine-proline-alanine (NPA) amino acid motifs i n the two halves of the protein. We recently demonstrated that y-TIP, one of the MIP homologs found i n the vacuolar membrane of plant cells, is an aquaporin or water channel protein (C. Maurel, J. Reizer, 1.1. Schroeder, M.J. Chrispeels [1993] EMBO J 12: 2241-2247). RD28, another MIP homolog in Arabidopsis thaliana, was first identified as being encoded by a turgor-responsive transcript. To find out if RD28 is a water channel protein, rd28 cRNA was injected into Xenopus laevis oocytes. Expression of RD28 caused a 10-to 15-fold increase i n the osmotic water permeability of the oocytes, indicating that the protein creates water channels in the plasma membrane of the oocytes and is an aquaporin just like its homolog ?-TIP. Although RD28 has several cysteine residues, its activity is not inhibited by mercury, and in this respect it differs from y-TIP and all but one of the mammalian water channels that have been described. Introduction of a cysteine residue next to the second conserved NPA motif creates a mercury-sensitive water channel, suggesting that this conserved loop is critical to the activity of the protein. Antibodies directed at the C terminus of RD28 were used in combination with a two-phase partitioning method to demonstrate that RD28 is located i n the plasma membrane. The protein is present in leaves and roots of well-watered plants, suggesting that its presence i n plants does not require a specific desiccation regime. These results demonstrate that plant cells contain constitutively expressed aquaporins i n their plasma membranes (RD28), as well as i n their tonoplasts (?-TIP).The vacuolar membranes of plant cells and the plasma membranes of mammalian cells contain aquaporins, proteins that form water-selective channels (see Chrispeels and Maurel, 1994, for review). These 27-kD integral membrane proteins belong to a family of proteins that has cognates in mammals, yeasts, and bacteria; they are part of the larger MIP family (see Reizer et al., 1993, for a recent review). In plants, more than half a dozen derived amino acid sequences and/or proteins have been recently identified. Some of them are expressed in a tissue-specific manner, whereas others are induced by specific physiological conditions. For example, a-TIP is a seed-specific protein found in the tonoplasts of protein storage vacuoles (Johnson et al., 1990), tobRB7 mRNA is found in the roots of tobacco (Nicotiana tabacum) (Yamamoto et al., 1991;Opperman et al., 1994), NOD26 is specific to the peribacteroid membranes of soybean (Glycine max) nodules (Sandal and Marcker, 1988), and trg-31 (Guerrero et al., 1990;Guerrero and Crossland, 1993) and rd28 (Yamaguchi-Shinozaki et al., 1992) transcripts are induced by desiccation of pea (Pisum sativum) and Arabidopsis thaliana, respectively. Dip is expres...

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We have derived the genomic nucleotide sequence of an emerging virus, the Sugarcane yellow leaf virus (ScYLV), and shown that it produces one to two subgenomic RNAs. The family Luteoviridae currently includes the Luteovirus, Polerovirus, and Enamovirus genera. With the new ScYLV nucleotide sequence and existing Luteoviridae sequence information, we have utilized new phylogenetic and evolutionary methodologies to identify homologous regions of Luteoviridae genomes, which have statistically significant altered nucleotide substitution ratios and have produced a reconstructed phylogeny of the Luteoviridae. The data indicate that Pea enation mosaic virus-1 (PEMV-1), Soybean dwarf virus (SbDV), and ScYLV exhibit spatial phylogenetic variation (SPV) consistent with recombination events that have occurred between poleroviral and luteoviral ancestors, after the divergence of these two progenitor groups. The reconstructed phylogeny confirms a contention that a continuum in the derived sequence evolution of the Luteoviridae has been established by intrafamilial as well as extrafamilial RNA recombination and expands the database of recombinant Luteoviridae genomes that are currently needed to resolve better defined means for generic discrimination in the Luteoviridae (D'Arcy, C. J. and Mayo, M. 1997. Arch. Virol. 142, 1285-1287). The analyses of the nucleotide substitution ratios from a nucleotide alignment of Luteoviridae genomes substantiates the hypothesis that hot spots for RNA recombination in this virus family are associated with the known sites for the transcription of subgenomic RNAs (Miller et al. 1995. Crit. Rev. Plant Sci. 14, 179-211), and provides new information that might be utilized to better design more effective means to generate transgene-mediated host resistance.

Development of CRISPR/Cas9 transient gene editing screening tools in plant biology has been hindered by difficulty of delivering high quantities of biologically active single guide RNAs (sgRNAs). Furthermore, it has been largely accepted that in vivo generated sgRNAs need to be devoid of extraneous nucleotides, which has limited sgRNA expression by delivery vectors. Here, we increased cellular concentrations of sgRNA by transiently delivering sgRNAs using a -derived vector (TRBO) designed with 5' and 3' sgRNA proximal nucleotide-processing capabilities. To demonstrate proof-of-principle, we used the TRBO-sgRNA delivery platform to target GFP in (16c) plants, and gene editing was accompanied by loss of GFP expression. Surprisingly, indel (insertions and deletions) percentages averaged nearly 70% within 7 d postinoculation using the TRBO-sgRNA constructs, which retained 5' nucleotide overhangs. In contrast, and in accordance with current models, in vitro Cas9 cleavage assays only edited DNA when 5' sgRNA nucleotide overhangs were removed, suggesting a novel processing mechanism is occurring in planta. Since the Cas9/TRBO-sgRNA platform demonstrated sgRNA flexibility, we targeted the paralogs with one sgRNA and also multiplexed two sgRNAs using a single TRBO construct, resulting in indels in three genes. TRBO-mediated expression of an RNA transcript consisting of an sgRNA adjoining a GFP protein coding region produced indels and viral-based GFP overexpression. In conclusion, multiplexed delivery of sgRNAs using the TRBO system offers flexibility for gene expression and editing and uncovered novel aspects of CRISPR/Cas9 biology.

RNA-mediated, posttranscriptional gene silencing has been determined as the molecular mechanism underlying transgenic virus resistance in many plant virus-dicot host plant systems. In this paper we show that transgenic virus resistance in sugarcane (Saccharum spp. hybrid) is based on posttranscriptional gene silencing. The resistance is derived from an untranslatable form of the sorghum mosaic potyvirus strain SCH coat protein (CP) gene. Transgenic sugarcane plants challenged with sorghum mosaic potyvirus strain SCH had phenotypes that ranged from fully susceptible to completely resistant, and a recovery phenotype was also observed. Clones derived from the same transformation event or obtained after vegetative propagation could display different levels of virus resistance, suggesting the involvement of a quantitative component in the resistance response. Most resistant plants displayed low or undetectable steady-state CP transgene mRNA levels, although nuclear transcription rates were high. Increased DNA methylation was observed in the transcribed region of the CP transgenes in most of these plants. Collectively, these characteristics indicate that an RNA-mediated, homology-dependent mechanism is at the base of the virus resistance. This work extends posttranscriptional gene silencing and homology-dependent virus resistance, so far observed only in dicots, to an agronomically important, polyploid monocot.Sugarcane (Saccharum spp. hybrid) ranks among the world's top 10 food crops and annually provides 60% to 70% of the sugar produced worldwide (Sugar and Sweetener Situation and Outlook Yearbook, 1997). Modern commercial sugarcane cultivars are interspecific hybrids derived from crosses of noble sugarcane, i.e. Saccharum officinarum L. (2n ϭ 70-122). Crosses are most often made with Saccharum spontaneum L. (2n ϭ 36-128), sometimes with Saccharum barberi (2n ϭ 60-140) or Saccharum sinense (2n ϭ 104-128), and rarely with Saccharum robustum (2n ϭ 66-170) (Irvine, 1999). Sugarcane cultivars have ploidy levels that range from 5ϫ to 14ϫ (ϫ ϭ 5, 6, 8, 10, 12, or 14) and chromosomal mosaicism has been reported (Burner and Legendre, 1994, and refs. therein). The genetic complexity and low fertility of sugarcane render traditional breeding laborious and make it a prime candidate for improvement through genetic engineering.Transgenic sugarcane plants have been obtained via particle gun bombardment of embryogenic callus (Bower and Birch, 1992; Gallo-Meagher and Irvine, 1996) and via electroporation of cells derived from embryogenic callus (Arencibia et al., 1995). Unlike many other members of the Poaceae in which regeneration is restricted to certain genotypes, most sugarcane cultivars tested to date have yielded regenerable calli. Therefore, introducing specific genetic improvements, such as virus resistance, directly into elite sugarcane varieties is a realistic goal.SCMV has a monopartite, positive-strand RNA genome (Shukla et al., 1994). Recent taxonomic studies have shown that the SCMV complex comprises four or five ...

The Sugarcane yellow leaf virus (SCYLV) P0, a member of the highly heterologous proteins of poleroviruses, is a suppressor of posttranscriptional gene silencing (PTGS) and has additional activities not seen in other P0 proteins. The P0 protein in previously tested poleroviruses (Beet western yellows virus and Cucurbit aphid-borne yellows virus), suppresses local, but not systemic, PTGS induced by both sense GFP and inverted repeat GF using its F-box-like domain to mediate destabilization of the Argonaute1 protein. We now report that the SCYLV P0 protein not only suppressed local PTGS induced by sense GFP and inverted repeat GF in Nicotiana benthamiana, but also triggered a dosage dependent cell death phenotype in infiltrated leaves and suppressed systemic sense GFP-PTGS. Deletion of the first 15 N-terminal amino acid residues of SCYLV P0 abolished suppression of both local and systemic PTGS and the induction of cell death. In contrast, only systemic PTGS and cell death were lost when the 15 C-terminal amino acid residues were deleted. We conclude that the 15 C-terminal amino acid residue region of SCYLV P0 is necessary for suppressing systemic PTGS and inducing cell death, but is not required for suppression of local PTGS.

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The common bean, Phaseolus vulgaris, contains a family of defense proteins that comprises phytohemagglutinin (PHA), arcelin, and alpha-amylase inhibitor (alpha AI). Here we report eight new derived amino acid sequences of genes in this family obtained with either the polymerase chain reaction using genomic DNA, or by screening cDNA libraries made with RNA from developing beans. These new sequences are: two alpha AI sequences and arcelin-4 obtained from a wild accession of P. vulgaris that is resistant to the Mexican bean weevil (Zabrotes subfasciatus) and the bean weevil (Acanthoscelides obtectus); an alpha AI sequence from the related species P. acutifolius (tepary bean); a PHA and an arcelin-like sequence from P. acutifolius; an alpha AI-like sequence from P. maculatus; and a PHA sequence from an arcelin-5 type P. vulgaris. A dendrogram of 16 sequences shows that they fall into the three identified groups: phytohemagglutinins, arcelins and alpha AIs. A comparison of these derived amino acid sequences indicates that one of the four amino acid residues that is conserved in all legume lectins and is required for carbohydrate binding is absent from all the arcelins; two of the four conserved residues needed for carbohydrate binding are missing from all the alpha AIs. Proteolytic processing at an Asn-Ser site is required for the activation of alpha AI, and this site is present in all alpha AI-like sequences; this processing site is also found at the same position in certain arcelins, which are not proteolytically processed. The presence of this site is therefore not sufficient for processing to occur.