Rice scutellum induces Agrobacterium tumefaciens vir genes and T-strand generation

For successful transformation of a plant by Agrobacterium tumefaciens it is essential that the explant used in cocultivation has the ability to induce Agrobacterium tumour-inducing (Ti) plasmid virulence (vir) genes. Here we report a significant variation in different tissues of Indica rice (Oryza sativa L. cv. Co43) in their ability to induce Agrobacterium tumefaciens vir genes and T-strand generation, using explants preincubated in liquid Murashige and Skoog (MS) medium. An analysis of rice leaf segments revealed that they neither induced vir genes nor inhibited vir gene induction. Of different parts of rice plants of different ages analysed only scutellum from four-day old rice seedlings induced vir genes and generation of T-strands. We observed that the physical presence of preincubated scutella is required for vir gene induction. Conditioned medium from which preincubated scutella were removed did not induce the vir genes. Scutellum-derived calli, cultured for 25 days on medium containing 2,4-D, also induced virE to an appreciable level. These results suggest that scutellum and scutellum-derived calli may be the most susceptible tissues of rice for Agrobacterium-mediated transformation.     

An In Silico Evaluation of Molecular Interaction Between Antimicrobial Peptide Subtilosin A of Bacillus subtilis with Virulent Proteins of Aeromonas hydrophila

International Journal of Peptide Research and Therapeutics - Subtilosin A, a cyclic peptide from Bacillus subtilis is known for its antimicrobial activity against a diverse range of bacteria....     

A second T-region of the soybean-supervirulent chrysopine-type Ti plasmid pTiChry5, and construction of a fully disarmed vir helper plasmid

Agrobacterium tumefaciens Chry5, which is particularly virulent on soybeans, induces tumors that produce a family of Amadori-type opines that includes deoxyfructosyl glutamine (Dfg) and its lactone, chrysopine (Chy). Cosmid clones mapping to the right of the known oncogenic T-region of pTiChry5 conferred Amadori opine production on tumors induced by the nopaline strain C58. Sequence analysis of DNA held in common among these cosmids identified two 25-bp, direct repeats flanking an 8.5-kb segment of pTiChry5. These probable border sequences are closely related to those of other known T-regions and define a second T-region of pTiChry5, called T-right (TR), that confers production of the Amadoriopines. The oncogenic T-left region (TL) was located precisely by identifying and sequencing the likely border repeats defining this segment. The two T-regions are separated by approximately 15 kb of plasmid DNA. Based on these results, we predicted that pKYRT1, a vir helper plasmid derived from pTiChry5, still contains all of TR and the leftmost 9 kb of TL. Consistent with this hypothesis, transgenic Arabidopsis thaliana plants selected for with a marker encoded by a binary plasmid following transformation with KYRT1 co-inherited production of the Amadori opines at high frequency. All opine-positive transgenic plants also contained TR-DNA, while those plants that lacked TR-DNA failed to produce the opines. Moreover, A. thaliana infected with KYRT1 in which an nptII gene driven by the 35S promoter of Cauliflower mosaic virus was inserted directly into the vir helper plasmid yielded kanamycin-resistant transformants at a low but detectable frequency. These results demonstrate that pKYRT1 is not disarmed, and can transfer Ti plasmid DNA to plants. A new vir helper plasmid was constructed from pTiChry5 by two rounds of sacB-mediated selection for deletion events. This plasmid, called pKPSF2, lacks both of the known T-regions and their borders. pKPSF2 failed to transfer Ti plasmid DNA to plants, but mobilized the T-region of a binary plasmid at an efficiency indistinguishable from those of pKYRT1 and the nopaline-type vir helper plasmid pMP90.     

Tobacco mosaic virus 126-kDa protein increases the susceptibility of Nicotiana tabacum to other viruses and its dosage affects virus-induced gene silencing

The Tobacco mosaic virus (TMV) 126-kDa protein is a suppressor of RNA silencing previously shown to delay the silencing of transgenes in Nicotiana tabacum and N. benthamiana. Here, we demonstrate that expression of a 126-kDa protein-green fluorescent protein (GFP) fusion (126-GFP) in N. tabacum increases susceptibility to a broad assortment of viruses, including Alfalfa mosaic virus, Brome mosaic virus, Tobacco rattle virus (TRV), and Potato virus X. Given its ability to enhance TRV infection in tobacco, we tested the effect of 126-GFP expression on TRV-mediated virus-induced gene silencing (VIGS) and demonstrate that this protein can enhance silencing phenotypes. To explain these results, we examined the poorly understood effect of suppressor dosage on the VIGS response and demonstrated that enhanced VIGS corresponds to the presence of low levels of suppressor protein. A mutant version of the 126-kDa protein, inhibited in its ability to suppress silencing, had a minimal effect on VIGS, suggesting that the suppressor activity of the 126-kDa protein is indeed responsible for the observed dosage effects. These findings illustrate the sensitivity of host plants to relatively small changes in suppressor dosage and have implications for those interested in enhancing silencing phenotypes in tobacco and other species through VIGS.     

The Cauliflower Mosaic Virus Protein P6 Forms Motile Inclusions That Traffic along Actin Microfilaments and Stabilize Microtubules

The gene VI product (P6) of Cauliflower mosaic virus (CaMV) is a multifunctional protein known to be a major component of cytoplasmic inclusion bodies formed during CaMV infection. Although these inclusions are known to contain virions and are thought to be sites of translation from the CaMV 35S polycistronic RNA intermediate, the precise role of these bodies in the CaMV infection cycle remains unclear. Here, we examine the functionality and intracellular location of a fusion between P6 and GFP (P6-GFP). We initially show that the ability of P6-GFP to transactivate translation is comparable to unmodified P6. Consequently, our work has direct application for the large body of literature in which P6 has been expressed ectopically and its functions characterized. We subsequently found that P6-GFP forms highly motile cytoplasmic inclusion bodies and revealed through fluorescence colocalization studies that these P6-GFP bodies associate with the actin/endoplasmic reticulum network as well as microtubules. We demonstrate that while P6-GFP inclusions traffic along microfilaments, those associated with microtubules appear stationary. Additionally, inhibitor studies reveal that the intracellular movement of P6-GFP inclusions is sensitive to the actin inhibitor, latrunculin B, which also inhibits the formation of local lesions by CaMV in Nicotiana edwardsonii leaves. The motility of P6 along microfilaments represents an entirely new property for this protein, and these results imply a role for P6 in intracellular and cell-to-cell movement of CaMV.Cauliflower mosaic virus (CaMV), the type member of the genus Caulimovirus, has a circular double-stranded DNA genome known to encode six open reading frames (ORFs). The gene product of ORF VI (P6) is a multifunctional protein whose ascribed functions have increased in number since its initial characterization over 20 years ago. P6 was originally described as the most abundant CaMV protein in infected plants (Odell and Howell, 1980) and was later shown to be the major constituent of amorphous, electron-dense inclusion bodies that are thought to be the sites of virion assembly (Fujisawa et al., 1967; Rubio-Huertos et al., 1968; Himmelbach et al., 1996; Cecchini et al., 1997). Indeed, despite the detection of other viral proteins in CaMV inclusions, the P6 protein on its own is capable of forming inclusion bodies (Cecchini et al., 1997; Li and Leisner, 2002; Haas et al., 2005).P6 is the major pathogenicity determinant for CaMV (Daubert et al., 1984; Baughman et al., 1988; Stratford and Covey, 1989; Zijlstra and Hohn, 1992) and was recently shown to be a suppressor of RNA silencing (Love et al., 2007). In addition, P6 also functions as an avirulence determinant, as it has been shown to be responsible for eliciting a hypersensitive response in Nicotiana edwardsonii and Datura stramonium, as well as nonnecrotic resistance in Nicotiana bigelovii and Arabidopsis (Arabidopsis thaliana) ectotype Tsu-O (Daubert et al., 1984; Schoelz et al., 1986; Wintermantel et al., 1993; Agama et al., 2002). The portion of the P6 protein recognized by plants is localized to the N-terminal third of the protein (Wintermantel et al., 1993; Palanichelvam et al., 2000; Agama et al., 2002). P6 also has a significant effect on plant metabolism, as it is responsible for down-regulating or inducing expression of several plant genes (Geri et al., 1999), including genes involved in ethylene signaling (Geri et al., 2004).Replication of CaMV involves the production of a polycistronic RNA intermediate, the 35S RNA, and P6 acts as a translational transactivator (TAV) by modifying the host translational machinery to allow for reinitiation of translation on this RNA (Ryabova et al., 2002). To carry out this function, the P6 protein physically interacts with the initiation factor eIF3 (Park et al., 2001), as well as ribosomal proteins L13 (Bureau et al., 2004), L18 (Leh et al., 2000), and L24 (Park et al., 2001). Finally, P6 is also a nucleocytoplasmic shuttle protein whose nuclear export is dependent upon a Leu-rich sequence near its N terminus, a region that is also involved in inclusion body formation (Li and Leisner, 2002; Haas et al., 2005). Although the precise role of the P6 protein''s nucleocytoplasmic shuttle function during infection remains to be elucidated, P6 does have the capacity to bind RNA (De Tapia et al., 1993; Cerritelli et al., 1998) and as such may act to control export of the 35S RNA from the nucleus to the cytoplasm, drawing the 35S RNA into the nascent P6 inclusion bodies where viral proteins are translated.Despite the recognized intracellular movement of P6 from cytoplasm to nucleus and the disparate cytoplasmic functions of this protein, factors controlling intracellular transport of P6 remain unknown. The cytoskeleton has been implicated in the intracellular trafficking of a number of plant viral proteins. For example, proteins encoded by several viruses have been found to colocalize with actin microfilaments, including the TGBp2 movement protein from Potato virus X (PVX), TGBp2 and TGBp3 from Potato mop-top virus, the Hsp70 homolog from Beet yellows virus, as well as both the movement (MP) and 126-kD proteins from Tobacco mosaic virus (TMV; McLean et al., 1995; Haupt et al., 2005; Ju et al., 2005; Liu et al., 2005; Prokhnevsky et al., 2005) In addition, inhibitor studies recently demonstrated that the intracellular trafficking of potato leafroll virus MP to the plasmodesmata (PD) is dependent upon an intact actin cytoskeleton (Vogel et al., 2007). Together, these studies suggest that the trafficking of viral proteins along actin filaments is a mechanism utilized by highly divergent RNA viruses.The only documented example of a plant viral protein found to colocalize with both microfilaments and microtubules in cells is the TMV MP (McLean et al., 1995; reviewed in Beachy and Heinlein, 2000; Lucas, 2006), which has been shown to associate with and stabilize microtubules and contains a motif thought to mimic the region of tubulin responsible for lateral junctions between microtubules (Boyko et al., 2000; Ashby et al., 2006). Interestingly, the CaMV gene II product (P2), an aphid transmission factor, was previously shown by immunoelectron microscopy to associate with microtubules in both insect and plant cells, although the significance of this interaction remains unclear (Blanc et al., 1996). In addition to these two viral proteins found to colocalize with microtubules in planta, the Hsp70 homolog from Beet yellows virus and the coat protein from PVX have both been shown to interact with microtubules in vitro (Karasev et al., 1992; Serazev et al., 2003). Evidence that the intracellular localization of grapevine fanleaf virus MP is disturbed by oryzalin, as well as the finding that the geminivirus replication protein AL1 interacts with a kinesin by yeast two-hybrid assay, may also indicate a potential association of these proteins with microtubules (Kong and Hanley-Bowdoin, 2002; Laporte et al., 2003).In this study, we utilize a fusion between the C terminus of P6 and GFP to visualize P6 inclusions in live cells. We demonstrate that the fusion of P6 with GFP does not interfere with its ability to act as a TAV. We further demonstrate that P6-GFP inclusion bodies move intracellularly and are associated with microtubules, actin microfilaments, and the endoplasmic reticulum (ER). Although P6-GFP inclusion bodies associated with microtubules appear stationary, we show that P6-GFP bodies can traffic along microfilaments and that this movement is severely reduced by treatment with the actin inhibitor latrunculin B (LatB). LatB treatment of N. edwardsonii leaves inhibits the formation of local lesions by CaMV, indicating the potential that P6 trafficking on microfilaments is necessary for CaMV cell-to-cell movement. Additionally, the association of P6-GFP inclusion bodies with microtubules prevents the disruption of microtubules by oryzalin, denoting a tight association between these two proteins. We discuss the potential role of P6 movement and cytoskeletal association in CaMV infection.     

Nuclear import of the capsid protein of tomato yellow leaf curl virus (TYLCV) in plant and insect cells

The tomato yellow leaf curl virus (TYLCV) found in Israel is a whitefly-transmitted monopartite geminivirus. Although geminiviruses have been found in the nuclei of phloem-associated cells, the mechanism of viral invasion is poorly understood. The possible role of the TYLCV capsid protein (CP), the only known component of the viral coat, in virus transport into the host cell nucleus was investigated by monitoring its specific nuclear accumulation in plant and insect cells. CP was fused to the β-glucuronidase (GUS) reporter enzyme to assay nuclear import in petunia protoplasts, and micro-injection of purified fluorescently labeled CP was used to examine its nuclear uptake in Drosophila embryos. Both assays demonstrated that TYLCV CP is transported into plant-and insect-cell nuclei by an active process of nuclear import via a nuclear localization signal (NLS)-specific pathway. Using the GUS assay and deletion analysis, the TYLCV CP NLS sequence was identified in the amino-terminus of the protein.     

Agroinfiltration of Cauliflower mosaic virus gene VI elicits hypersensitive response in Nicotiana species

Cauliflower mosaic virus strain W260 induces hypersensitive response (HR) in Nicotiana edwardsonii and systemic cell death in N. clevelandii. In contrast, the D4 strain of Cauliflower mosaic virus evades the host defenses in Nicotiana species; it induces chlorotic primary lesions and a systemic mosaic in both hosts. Previous studies with chimeric viruses had indicated that gene VI of W260 was responsible for elicitation of HR or cell death. To prove conclusively that W260 gene VI is responsible, we inserted gene VI of W260 and D4 into the Agrobacterium tumefaciens binary vector pKYLX7. Agroinfiltration of these constructs into the leaves of N. edwardsonii and N. clevelandii revealed that gene VI of W260 elicited HR in N. edwardsonii 4 to 5 days after infiltration and cell death in N. clevelandii approximately 9 to 12 days after infiltration. In contrast, gene VI of D4 did not elicit HR or cell death in either Nicotiana species. A frameshift mutation introduced into gene VI of W260 abolished its ability to elicit HR or cell death in both Nicotiana species, demonstrating that the elicitor is the gene VI protein.     

Silencing of the N family of resistance genes in Nicotiana edwardsonii compromises the hypersensitive response to tombusviruses

The nontarget effects associated with silencing of the N gene in Nicotiana edwardsonii, an amphidiploid species derived from N. glutinosa and N. clevelandii, have been characterized in this study. The N protein confers resistance to Tobacco mosaic virus (TMV), and is representative of a family of nucleotide-binding site leucine-rich repeat proteins present in N. glutinosa. Previous studies have shown that silencing of the N gene or of other plant genes associated with N-mediated defenses abolishes host resistance to TMV, and this effect can be measured through enhancements in movement or replication of TMV in the N-silenced plants. However, the nontarget effects of gene silencing have not been investigated thoroughly. Notably, are the functions of other resistance (R) genes also affected in experiments designed to silence the N gene? To investigate whether heterologous sequences could silence the N gene, we selected an R gene homolog from N. glutinosa that differed from the N gene by approximately 17%, created a hairpin transgene, and developed transgenic N. edwardsonii plants. Expression of this hairpin in the transgenic N. edwardsonii plants compromised the hypersensitive response to TMV, demonstrating that a single hairpin transgene could silence a block of R genes related by sequence similarity. We then investigated whether the response of N-silenced plants to other viruses would be altered, and found that the hypersensitive response triggered against the tombusviruses Tomato bushy stunt virus and Cymbidium ringspot virus also was compromised. This study indicates that a Tombusvirus R gene shares some homology with the N gene, which could facilitate the cloning of this gene.     

Population genetic structure of Penaeus monodon, in relation to monsoon current patterns in Southwest, East and Andaman coastal waters of India

The black tiger shrimp (Penaeus monodon), a commercially important penaeid species, is widely distributed across the Indo-Pacific region. Genetic diversity in P. monodon collected from eight geographical regions in Southwest, East and Andaman coastal waters of India (N = 418) was investigated using 10 polymorphic microsatellite loci. Average observed heterozygosity at sampled loci were high, ranging from 0.643 (Coromandel Coast) to 0.753 (South Andaman). Pairwise F_ST (ranged from 0.005 to 0.078) and R_ST (ranged from 0.005 to 0.171) estimates revealed surprisingly strong and statistically significant genetic structure among tiger shrimp populations. A synthetic map generated by multidimensional scaling shows an apparent cline in allele frequencies paralleling the roughly circular flow of surface currents in the Bay of Bengal. Significant heterozygote deficiencies were noted in most population samples at most loci. Andaman Island sites showed the highest diversity. Recognition of high genetic diversity and distinct population structuring of P. monodon in Indian seas has important implications for future domestication of this species in India, for two reasons: identification of the best wild founding stocks for aquaculture and, subsequently, the potential impacts of release of domesticates to the wild, either accidentally or deliberately (i.e. for stock enhancement).