Detection of self-complementary inverted repeats by single forward primer driven PCR

Inverted repeat gene structures designed for silencing functional genes have been widely used both in academic and applied research. The correct orientations of such structures are usually validated with restriction analysis and/or sequencing. We speculated that the inverted repeat nature of such constructs can be shown by a simple PCR reaction with a single forward primer. To test this hypothesis five different constructs were established from grapevine sequences in a hairpin-intron style silencing system. We were able to amplify the appropriate products in each case. Thus a forward-primed PCR alone may be sufficient to prove the inverted repeat nature of the desired constructs

Detection of self-complementary inverted repeats by single forward primer driven PCR

Inverted repeat gene structures designed for silencing functional genes have been widely used both in academic and applied research. The correct orientations of such structures are usually validated with restriction analysis and/or sequencing. We speculated that the inverted repeat nature of such constructs can be shown by a simple PCR reaction with a single forward primer. To test this hypothesis five different constructs were established from grapevine sequences in a hairpin-intron style silencing system. We were able to amplify the appropriate products in each case. Thus a forward-primed PCR alone may be sufficient to prove the inverted repeat nature of the desired constructs

Agrobacterium vitis strains lack tumorigenic ability on in vitro grown grapevine stem segments

Grapevine stem segments were cocultivated with three different Agrobacterium tumefaciens and three different A. vitis strains. A. tumefaciens strains induced tumors at variable frequencies, while A. vitis-infected stem segments never formed crown galls. The tumorous nature of tissues grown on hormone free medium was confirmed by opine assays. Bioinformatic and PCR analysis of the virulence regions of various A. tumefaciens and A. vitis Ti plasmids showed that virH2 and virK genes are common in A. tumefaciens but they are lacking from A. vitis. Thus virH2 and virK genes may be essential for grapevine stem segment transformation, but expression of certain T-DNA genes of A. vitis may also prevent the growth of transformed cells. Our data indicate that the tumorigenic ability of A. vitis is different on intact plant and on their explants, and that the specific host association of A. vitis on grapevine is probably determined by physiological and biochemical factors (e. g., better colonizing ability) rather than by its increased tumorigenic ability. Therefore it is not reasonable to develop „helper” plasmids for grapevine transformation from A. vitis pTis, unless their avirulence on in vitro explants is determined by T-DNA gene(s). Due to the inability of A. vitis to induce tumors on grapevine stem segments, the use of in vitro explant assays cannot be reliably used to select A. vitis resistant grapevine genotypes or transgenic lines

A ferritin szerepe a szőlő stressztűrő képességének fokozásában = The role of ferritin in enhancing the stress tolerance of grapevine

Embriogén kalluszt állítottuk elő Richter110 (alany) és Chardonnay (nemes) szőlő fajták portokajiból. Ezeket Medicago sativa ferritint (MsFerr) tartalmazó Agrobacterium vektorokkal transzformáltunk. A DNS beépülését genomikus PCR-rel ellenőriztük. Poliklonális ellenanyagot állítottunk elő, mellyel megállapítottuk, hogy a transzláció magas szintű volt, a transzkriptumból nagy mennyiségű, megfelelően processzált fehérjetermék keletkezett. A transzgenikus szőlő növények elkészültéig dohányban teszteltük az MsFerr gént tartalmazó konstrukciókat. A teljes növényeken végzett UV-B kezelés, ill. levélkorongok közvetlen oxidatív stresszre adott válaszai alapján megállapítottuk, hogy az MsFerr növények toleránsabbak voltak, mint a nem expresszáló kontrollok. Az MsFerr Richter 110 növények regenerálása, szelekciója és felszaporítása sikeres volt, az MsFerr Chardonnay növények felszaporítása azonban hajtásnövekedési problémák miatt nem sikerült. Három MsFerr Richter 110 vonalat vizsgáltunk. Gyökér stresszként fás szárú dugványok tápoldatához hidrogénkarbonátot adtunk, ami klorotikus és levél száradási tünetek okozott Ebben nem kaptunk lényeges különbséget a transzgenikus és transzformálatlan növények között. Ezzel szemben, a leveleket érő hatásokkal: paraquat, NaCl só-stressz és tBHP indukált lipid peroxidációval szemben az MsFerr expresszáló vonalak toleránsabbak voltak (kisebb stressz-indukált fotoszintézis csökkenést mutattak) mint a transzformálatlanok. | Embryogenic calli were started from anthers of Richter 110 (rootstock) and Chardonnay (scion) grapevine and transformed with Agrobacterium harbouring Medicago sativa ferritin gene (MsFerr). Genomic PCR and protein immunoblotting using a polyclonal antibody confirmed that the transcription and processing of MsFerr was successful. Regeneration, selection and propagation of MsFerr Richter 110 plants was successful, but transformed MsFerr Chardonnay plants did not grow sufficiently. Until transgenic grapevine plants became available, preliminary experiments were carried out with MsFerr expressing tobacco. UV-B irradiation of whole plants as well as treatments of leaf disks with various chemical elicitors showed that MsFerr plants were more stress tolerant than non-expressing controls. After regeneration and propagation, three transgenic MsFerr Richter 110 lines were tested. Roots of green cuttings were stressed by flooding and in this experiment transgenic plants did not show significantly higher tolerance to hypoxia/bicarbonate than non-transferred ones. Leaves, however, showed increased tolerance to paraquat, salt stress and tBHP induced lipid peroxidation: their photosynthesis was less affected by these stressors than those from non-transformed plants

Candidate plant gene homologues in grapevine involved in Agrobacterium transformation

Abstract The grapevine (Vitis vinifera) genome was analyzed in silico for homologues of plant genes involved in Agrobacterium transformation in Arabidopsis thaliana and Nicotiana spp. Grapevine homologues of the glucomannan 4-betamannosyltransferase 9 gene CslA-09 involved in bacterial attachment to the cell wall, homologues of reticulon-like proteins BTI1, 2, 3 and RAB8 GTPases, both involved in T-DNA transfer to the host cell, homologues of VirE2 interacting protein VIP1 that contributes to the targeting of T-DNA into the nucleus and to its integration, and homologues of the histone protein H2A, which promotes the expression of T-DNA encoded genes, were selected. Sequences homologous to the arabinogalactan-protein AtAGP17 were not found in the grape genome. Seventeen selected candidates were tested by semiquantitative RT-PCR analysis for changes in their expression levels upon inoculation with Agrobacterium tumefaciens C58. Of the tested homologues, the expression of VvRab8a, VvVip1a and two histone genes (VvHta2 and VvHta10) increased significantly, therefore we hypothesize that these might be involved in Agrobacterium transformation of V. vinifera.</jats:p

Quorum-sensing signal production by Agrobacterium vitis strains and their tumor-inducing and tartrate-catabolic plasmids

Agrobacterium vitis strains, their tumor-inducing (pTi) and tartrate utilization (pTr) plasmid transconjugants and grapevine tumors were analyzed for the presence of N-acyl-homoserine lactones (AHLs). All wild-type A. vitis strains produced long-chain signals. PCR analysis of the A. vitis long-chain AHL synthase gene, avsI, showed the predicted amplicon. Agrobacterium tumefaciens UBAPF2 harboring various A. vitis pTi plasmids produced N-(3-oxo-octanoyl)-l-homoserine lactone encoded also by pTis of A. tumefaciens. UBAPF2 transconjugants carrying pTrs except for pTrTm4 and pTrAB3, also produced an AHL. UBAPF2 transconjugants carrying pTrAT6, pTrAB4 and pTrRr4 or pTiNi1 produced two additional AHLs not observed in the corresponding wild-type strains. We also provide evidence for in situ production of AHLs in grapevine crown gall tumors of greenhouse and field origi

Culture media supplemented with inorganic salts improve the growth and viability of several bacterial strains

In order to improve growth and storage conditions for bacterial cultures, commonly used basic culture media, Luria-Bertani broth (LB) and glucose-yeast extract (GY) were tested along with their supplemented versions (LBS and GYS) containing a complex set of inorganic salts required for common physiological processes. The growth kinetics and viability of 15 representative strains were compared on LB/LBS or GY/GYS. Growth kinetics were examined during a 24 h period. Five out of 15 strains showed enhanced growth on LBS and GYS. Three strains showed very low viability (3 months or lower) both on the basic and salt-supplemented media. Six strains could be equally recovered after 6 or 12 months both from LB/LBS and from GY/GYS. Six of the tested 15 bacterial strains showed significantly better recovery rate on the inorganic-supplemented media LBS or GYS than on basic LB or GY. These results show that inorganic supplement of basic media may significantly improve the growth and viability of several bacterial strains

Culture media supplemented with inorganic salts improve the growth and viability of several bacterial strains

In order to improve growth and storage conditions for bacterial cultures, commonly used basic culture media, Luria-Bertani broth (LB) and glucose-yeast extract (GY) were tested along with their supplemented versions (LBS and GYS) containing a complex set of inorganic salts required for common physiological processes. The growth kinetics and viability of 15 representative strains were compared on LB/LBS or GY/GYS. Growth kinetics were examined during a 24 h period. Five out of 15 strains showed enhanced growth on LBS and GYS. Three strains showed very low viability (3 months or lower) both on the basic and salt-supplemented media. Six strains could be equally recovered after 6 or 12 months both from LB/LBS and from GY/GYS. Six of the tested 15 bacterial strains showed significantly better recovery rate on the inorganic-supplemented media LBS or GYS than on basic LB or GY. These results show that inorganic supplement of basic media may significantly improve the growth and viability of several bacterial strains