Trehalose Toxicity in Cuscuta reflexa: SUCROSE CONTENT DECREASES IN SHOOT TIPS UPON TREHALOSE FEEDING

Trehalose, an α,α-diglucoside, induced a rapid blackening and death of shoot tips of Cuscuta reflexa (dodder) cultured in vitro. The onset of toxic symptom was delayed if any of the several sugars which support the in vitro growth of Cuscuta was supplied with trehalose. The rate of trehalose uptake or its accumulation in the tissue was not affected by sugar cofeeding. The levels of total and reducing sugars declined appreciably in the trehalose-fed shoot tip explants compared to control tissue cultured in absence of a carbon source. This was not due to an increased rate of respiration of the trehalose-treated tissue. In shoot tips cultured in presence of both trehalose and sucrose, the decline in total and reducing sugars was curtailed. There was a marked fall in the level of sucrose; and invertase activity was higher in trehalose-fed shoot tips. The incorporation of label from [¹⁴C]glucose into sucrose in the shoot tip explant was reduced as early as 12 h of trehalose feeding. The results suggest that increased utilization of sucrose as well as an inhibition of its synthesis contribute to the drastic fall in the sucrose content upon trehalose feeding.     

Efficient vir gene induction in Agrobacterium tumefaciens requires virA, virG, and vir box from the same Ti plasmid

The vir genes of octopine, nopaline, and L,L-succinamopine Ti plasmids exhibit structural and functional similarities. However, we observed differences in the interactions between octopine and nopaline vir components. The induction of an octopine virE(A6)::lacZ fusion (pSM358cd) was 2.3-fold higher in an octopine strain (A348) than in a nopaline strain (C58). Supplementation of the octopine virG(A6) in a nopaline strain with pSM358 did not completely restore virE(A6) induction. However, addition of the octopine virA(A6) to the above strain increased virE(A6) induction to a level almost comparable to that in octopine strains. In a reciprocal analysis, the induction of a nopaline virE(C58)::cat fusion (pUCD1553) was two- to threefold higher in nopaline (C58 and T37) strains than in octopine (A348 and Ach5) and L,L-succinamopine (A281) strains. Supplementation of nopaline virA(C58) and virG(C58) in an octopine strain (A348) harboring pUCD1553 increased induction levels of virE(C58)::cat fusion to a level comparable to that in a nopaline strain (C58). Our results suggest that octopine and L,L-succinamopine VirG proteins induce the octopine virE(A6) more efficiently than they do the nopaline virE(C58). Conversely, the nopaline VirG protein induces the nopaline virE(C58) more efficiently than it does the octopine virE(A6). The ability of Bo542 virG to bring about supervirulence in tobacco is observed for an octopine vir helper (LBA4404) but not for a nopaline vir helper (PMP90). Our analyses reveal that quantitative differences exist in the interactions between VirG and vir boxes of different Ti plasmids. Efficient vir gene induction in octopine and nopaline strains requires virA, virG, and vir boxes from the respective Ti plasmids.     

<Emphasis Type="Italic">Agrobacterium</Emphasis> T-DNA insertion in the rice <Emphasis Type="Italic">DWARF SHOOT AND DEFECTIVE PANICLE1</Emphasis> (<Emphasis Type="Italic">DSDP1</Emphasis>) gene causes a severe dwarf phenotype,reduces plant vigour,and affects seed germination

We report a new T-DNA-tagged rice plant chi7, which displays a severe dwarf phenotype, reduced plant vigour, and impaired panicle development in the homozygous state. By chromosome walking, T-DNA integration was mapped in chromosome 2, 1054-bp upstream of the translation start site of a gene (Os02g0820400), which we designate as DWARF SHOOT AND DEFECTIVE PANICLE1 (DSDP1). DSDP1 expression was unexpectedly higher in the homozygous mutant leaves than in the hemizygous mutant and control plant leaves. Mutant dsdp1 seeds, stored for 24 weeks, failed to germinate in soil. The growth vigour of the dsdp1 mutant reduced with increasing seed storage period. The dsdp1 mutant plants, grown in vitro on MS medium, formed short, stout, and ageotropic roots with lesser number of root hairs. The findings suggest that DSDP1 may function as a negative regulator of many developmental processes in rice.     

The ORF3 protein of porcine circovirus type 2 interacts with porcine ubiquitin E3 ligase Pirh2 and facilitates p53 expression in viral infection

Porcine circovirus type 2 (PCV2) is the primary causative agent of an emerging swine disease, postweaning multisystemic wasting syndrome. We previously showed that a newly identified protein, ORF3, plays a major role in virus-induced apoptosis and is involved in viral pathogenesis in vitro and in vivo. To characterize the role of the ORF3 protein in modulation of cellular function, a yeast two-hybrid system was used to screen a porcine cDNA library to find its interacting partner. We have isolated and characterized pPirh2 (for "porcine p53-induced RING-H2"), an E3 ubiquitin ligase, which specifically interacts with the ORF3 protein of PCV2. This interaction was further confirmed when the ORF3 protein coimmunoprecipitated with and colocalized to pPirh2 in PK15 cells. The ORF3 protein has been found to interact with the p53 binding domain of pPirh2 in yeast cells. Expression of the protein results in less pPirh2 expression in PCV2-infected cells. Furthermore, increases in p53 expression were observed in PCV2-infected and ORF3 (alone)-transfected cells. Phosphorylation of p53 at Ser-46, which is related to p53-induced apoptosis, was also time-dependently activated in PCV-infected and ORF3-transfected cells. Taken together, our results show that the PCV2 ORF3 protein specifically interacts with pPirh2 and inhibits its stabilization; this may lead to increasing p53 expression, resulting in apoptosis.     

A novel T-DNA integration in rice involving two interchromosomal translocations

Key message

A male sterile transgenic rice plant TC-19 harboured a novel T-DNA integration in chromosome 8 with two interchromosomal translocations of 6.55 kb chromosome 3 and 29.8 kb chromosome 9 segments.

Abstract

We report a complex Agrobacterium T-DNA integration in rice (Oryza sativa) associated with two interchromosomal translocations. The T-DNA-tagged rice mutant TC-19, which harboured a single copy of the T-DNA, displayed male sterile phenotype in the homozygous condition. Analysis of the junctions between the T-DNA ends and the rice genome by genome walking showed that the right border is flanked by a chromosome 3 sequence and the left border is flanked by a chromosome 9 sequence. Upon further walking on chromosome 3, a chromosome 3/chromosome 8 fusion was detected. Genome walking from the opposite end of the chromosome 8 break point revealed a chromosome 8/chromosome 9 fusion. Our findings revealed that the T-DNA, together with a 6.55-kb region of chromosome 3 and a 29.8-kb region of chromosome 9, was translocated to chromosome 8. Southern blot analysis of the homozygous TC-19 mutant revealed that the native sequences of chromosome 3 and 9 were restored but the disruption of chromosome 8 in the first intron of the gene Os08g0152500 was not restored. The integration of the complex T-DNA in chromosome 8 caused male sterility.     

Octopine- and Nopaline-Inducible Proteins in Agrobacterium tumefaciens Are Also Induced by Arginine

Octopine induced the synthesis of 83, 76, 62, 58, 44, 42, 31, and 22 kDa proteins in Agrobacterium tumefaciens strains harboring the tumor-inducing (Ti) plasmids pTiA6 and pTiAch5. Nopaline induced the synthesis of 83, 76, 62, 58, 56, 44, 42, 31, and 22 kDa proteins in A. tumefaciens strains harboring the Ti plasmids pTiC58 and pTiT37. The molecular masses of proteins induced by octopine and nopaline were very similar. In accordance with the ‘opine concept’, octopine and nopaline were found to induce protein synthesis only in strains harboring the respective Ti plasmids. Arginine, a common catabolic product of octopine and nopaline, induced the synthesis of most of the proteins induced by the two opines. Our results show that only the initial step(s) of octopine and nopaline catabolism are induced by specific opines in the respective strains. The subsequent steps are likely to be regulated by arginine in both strains. Received: 5 January 1996 / Accepted: 21 February 1996