RNA interference-mediated resistance to maize dwarf mosaic virus

Maize dwarf mosaic virus (MDMV) is a widespread pathogen that causes serious yield loss to maize crops. A hairpin RNA expression vector was constructed herein to overcome the low efficiency of cultural protection against MDMV and to improve the MDMV resistance mediated by a shorter transgenic inverted-repeat sequence. This expression vector contained a 451 bp inverted-repeat sequence, homologous to the protease gene (P1) of MDMV. It was used for the Agrobacterium tumefaciens-mediated transformation of maize calli induced from a susceptible inbred line. A total of 17 T₂ transgenic lines were identified by both specific PCR amplification and Southern blot hybridization. Of these lines, 15 were evaluated for MDMV resistance in inoculation field trials under two environments. The relative replication levels of the P1 gene were analyzed by quantitative real-time (qRT)-PCR. Results demonstrated that all of the 15 T₂ lines showed an enhanced resistance to MDMV in comparison with that of the non-transformed parent line. Six lines were deemed to be ‘resistant’ with an average disease index below 25 %, which was not significantly different from that of the resistant control. The relative replication levels of the virus gene were significantly reduced in these resistant T₂ transgenic lines. The efficiency of virus gene silencing was directly related to the transgene copy numbers presented in these transgenic lines.     

Control of Mn status and infection rate by genotype of both host and pathogen in the wheat take-all interaction

Take-all is a world-wide root-rotting disease of cereals. The causal organism of take-all of wheat is the soil-borne fungus Gaeumannomyces graminis var tritici (Ggt). No resistance to take-all, worthy of inclusion in a plant breeding programme, has been discovered in wheat but the severity of take-all is increased in host plants whose tissues are deficient for manganese (Mn). Take-all of wheat will be decreased by all techniques which lift Mn concentrations in shoots and roots of Mn-deficient hosts to adequate levels. Wheat seedlings were grown in a Mn-deficient calcareous sand in small pots and inoculated with four field isolates of Ggt. Infection by three virulent isolates was increased under conditions which were Mn deficient for the wheat host but infection by a weakly virulent isolate, already low, was further decreased. Only the three virulent isolates caused visible oxidation of Mn in vitro. The sensitivity of Ggt isolates to manganous ions in vitro did not explain the extent of infection they caused on wheat hosts. In a similar experiment four Australian wheat genotypes were grown in the same Mn-deficient calcareous sand and inoculated with one virulent isolate of Ggt. Two genotypes were inefficient at taking up manganese and were very susceptible to take-all, one was very efficient at taking up manganese and was resistant to take-all, and the fourth genotype was intermediate for both characters. All genotypes were equally resistant under Mn-adequate conditions.     

Development of marker-free transgenic lettuce resistant to <Emphasis Type="Italic">Mirafiori lettuce big</Emphasis>-<Emphasis Type="Italic">vein virus</Emphasis>

Lettuce big-vein disease caused by Mirafiori lettuce big-vein virus (MLBVV) is found in major lettuce production areas worldwide, but highly resistant cultivars have not yet been developed. To produce MLBVV-resistant marker-free transgenic lettuce that would have a transgene with a promoter and terminator of lettuce origin, we constructed a two T-DNA binary vector, in which the first T-DNA contained the selectable marker gene neomycin phosphotransferase II, and the second T-DNA contained the lettuce ubiquitin gene promoter and terminator and inverted repeats of the coat protein (CP) gene of MLBVV. This vector was introduced into lettuce cultivars ‘Watson’ and ‘Fuyuhikari’ by Agrobacterium tumefaciens-mediated transformation. Regenerated plants (T₀ generation) that were CP gene-positive by PCR analysis were self-pollinated, and 312 T₁ lines were analyzed for resistance to MLBVV. Virus-negative plants were checked for the CP gene and the marker gene, and nine lines were obtained which were marker-free and resistant to MLBVV. Southern blot analysis showed that three of the nine lines had two copies of the CP gene, whereas six lines had a single copy and were used for further analysis. Small interfering RNAs, which are indicative of RNA silencing, were detected in all six lines. MLBVV infection was inhibited in all six lines in resistance tests performed in a growth chamber and a greenhouse, resulting in a high degree of resistance to lettuce big-vein disease. Transgenic lettuce lines produced in this study could be used as resistant cultivars or parental lines for breeding.     

Enhanced resistance to stripe rust disease in transgenic wheat expressing the rice chitinase gene RC24

Stripe rust is a devastating fungal disease of wheat worldwide which is primarily caused by Puccinia striiformis f. sp tritici. Transgenic wheat (Triticum aestivum L.) expressing rice class chitinase gene RC24 were developed by particle bombardment of immature embryos and tested for resistance to Puccinia striiformis f.sp tritici. under greenhouse and field conditions. Putative transformants were selected on kanamycin-containing media. Polymease chain reaction indicated that RC24 was transferred into 17 transformants obtained from bombardment of 1,684 immature embryos. Integration of RC24 was confirmed by Southern blot with a RC24-labeled probe and expression of RC24 was verified by RT-PCR. Nine transgenic T₁ lines exhibited enhanced resistance to stripe rust infection with lines XN8 and BF4 showing the highest level of resistance. Southern blot hybridization confirmed the stable inheritance of RC24 in transgenic T₁ plants. Resistance to stripe rust in transgenic T₂ and T₃ XN8 and BF4 plants was confirmed over two consecutive years in the field. Increased yield (27–36 %) was recorded for transgenic T₂ and T₃ XN8 and BF4 plants compared to controls. These results suggest that rice class I chitinase RC24 can be used to engineer stripe rust resistance in wheat.     

Overexpression of the Gossypium barbadense Actin-Depolymerizing Factor 1 Gene Mediates Biological Changes in Transgenic Tobacco

The function of a member of the actin-depolymerizing factor family from Gossypium barbadense, GbADF1, was investigated. Tobacco (Nicotiana tabacum) lines expressing GbADF1 were produced by Agrobacterium-mediated transformation. Southern and northern blot analyses showed that GbADF1 was successfully incorporated as a single copy into the tobacco genome and stably expressed in three lines of T₁ transgenic tobacco plants. Biological changes were detected in these transgenic lines, wherein GbADF1 transgenic seedlings exhibited shorter hypocotyls along with fewer root hairs than those of control plants. Moreover, guard cells of leaves of the transgenic plants were induced to close stomata, while flowering was delayed 5 days in T₁ lines compared to those of empty vector transgenic control plants. Segregation of GbADF1 in the T₂ generation fits the expected 3:1 ratio corresponding to a single dominant gene. Subsequently, GbADF1 was fused to the green fluorescent protein gene to generate a fusion expression vector. Transient expression analysis indicated that this fusion protein was localized in the nucleus and cytoskeleton of epidermal cells of onion. These results suggest that actin-depolymerizing factor 1 gene from G. barbadense plays an important role in the process of plant cell morphogenesis.     

Over-expression of snakin-2 and extensin-like protein genes restricts pathogen invasiveness and enhances tolerance to Clavibacter michiganensis subsp. michiganensis in transgenic tomato (Solanum lycopersicum)

Two tomato proteins were evaluated by over-expression in transgenic tomato for their ability to confer resistance to Clavibacter michiganensis subsp. michiganensis (Cmm). Snakin-2 (SN2) is a cysteine-rich peptide with broad-spectrum antimicrobial activity in vitro while extensin-like protein (ELP) is a major cell-wall hydroxyproline-rich glycoprotein linked with plant response to pathogen attack and wounding. Tomato plants, cultivar Mountain Fresh, were transformed via Agrobacterium tumefaciens harboring a binary vector for expression of the full-length SN2 gene or ELP cDNA under the regulation of the CaMV 35S promoter. Molecular characterization of PCR-positive putative T₀ transgenic plants by Northern analysis revealed constitutive over-expression of SN2 and ELP mRNA. Junction fragment analysis by Southern blot showed that three of the four SN2 over-expressing T₀ lines had single copies of complete T-DNAs while the other line had two complete T-DNA copies. All four ELP over-expressing T₀ lines had a single copy T-DNA insertion. Semi-quantitative RT-PCR analysis of T₁ plants revealed constitutive over-expression of SN2 and ELP. Transgenic lines that accumulated high levels of SN2 or ELP mRNA showed enhanced tolerance to Cmm resulting in a significant delay in the development of wilt symptoms and a reduction in the size of canker lesions compared to non-transformed control plants. Furthermore, in transgenic lines over-expressing SN2 or ELP bacterial populations were significantly lower (100–10,000-fold) than in non-transformed control plants. These results demonstrate that SN2 and ELP over-expression limits Cmm invasiveness suggesting potential in vivo antibacterial activity and possible biotechnological application for these two defense proteins.     

Transgenic tomato plants expressing a wheat endochitinase gene demonstrate enhanced resistance to <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis>

Various chitinases have been shown to inhibit the growth of fungal pathogens in in vitro as well as in planta conditions. chi194, a wheat chitinases gene encoding a 33-kDa chitinase protein, was overexpressed in tomato plants (cv. Pusa Ruby) under the control of maize ubiquitin 1 promoter. The integration of transgene in tomato plants was confirmed with polymerase chain reaction (PCR) and Southern blot analysis. The inheritance of the transgene in T₁ and T₂ generations were shown by molecular analysis and the hygromycin sensitivity test. The broad range of chitinase activity was observed among the transgenic lines in T₀ and a similar range was retained in the T₁ and T₂ generations. Most importantly, the transgenic tomato lines with high chitinase activity were found to be highly resistant to the fungal pathogen Fusarium oxysporum f. sp. lycopersici. Thus, the results demonstrated that the expression of the wheat endochitinase chi194 in tomato plants confers resistance against Fusarium wilt disease caused by the fungal pathogen Fusarium oxysporum f. sp. lycopersici.     

Transgenic <Emphasis Type="Italic">indica</Emphasis> rice cultivar ‘Swarna’ expressing a potato chymotrypsin inhibitor <Emphasis Type="Italic">pin2</Emphasis> gene show enhanced levels of resistance to yellow stem borer

Transgenic rice was developed from ‘Swarna’, the most popular indica rice cultivar (Oryza sativa L.) in South East Asia, with a potato chymotrypsin inhibitor gene (pin2) through Agrobacterium-mediated transformation. Four out of nine primary transgenic plants had a single-copy T-DNA insertion while other five plants had two copies. Mendelian pattern of inheritance of the transgene (pin2) was observed in the T₁ generation progeny plants. Whole plant bioassays conducted at both vegetative and reproductive stages and cut stem assays showed enhanced levels of resistance of transgenic rice against yellow stem borer. The transgenic rice lines with plant derived proteinase inhibitor genes would develop into resistant cultivars to fit into resistance breeding strategies as an important component of integrated pest management in rice.     

Stability of sheath blight resistance in transgenic ASD16 rice lines expressing a rice <Emphasis Type="Italic">chi11</Emphasis> gene encoding chitinase

Development of transgenic plants by introducing defense genes is one of the strategies to engineer disease resistance. Transgenic ASD16 rice plants harbouring rice chitinase chi11 gene, belonging to a PR-3 group of defense gene conferring sheath blight (Rhizoctonia solani Kuhn) resistance, were used in this study. Three T₂ homozygous lines (ASD16-4-1-1, 5-1-1, and 6-1-1) were identified from seven putative (T₀) transgenic lines expressing chi11 using Western blotting analysis. The inheritance of sheath blight resistance in those lines was studied over generations. The stability of chi11 expression up to T₄ generation in all the three homozygous lines was proved by Western blot and the stability of sheath blight resistance in the homozygous lines was proved up to T₄ generation using detached leaf and intact leaf sheath assays. Among the three homozygous lines tested, ASD16-4-1-1 showed consistent results in all the generations and gave a better protection against the sheath blight pathogen than the other two lines.     

Agrobacterium-mediated transformation of cotton (Gossypium hirsutum L. cv. Zhongmian 35) using glyphosate as a selectable marker

The most economically significant Chinese cotton cultivar (Gossypium hirsutum L. cv. Zhongmian 35) was transformed via Agrobacterium tumefaciens-mediated DNA transfer. The aroA-M1 gene that confers resistance to the glyphosate was fused with a chloroplast-transit peptide of Arabidopsis thaliana 5-enolpyruvyl-3-phosphoshikimate synthase (ASP) and expressed in cotton plants under the control of a CaMV35S promoter. Transgenic plants were directly selected on medium containing glyphosate. Thirty-four independent transgenic lines were obtained after selection, giving a maximal 1.9% transformation frequency. The integration and expression of the aroA-M1 gene in T₀ plants and T₁ progeny were confirmed using DNA hybridization, Western blot and PCR techniques. An increased resistance of T₀ and T₁ transgenic plants towards glyphosate was also observed.     

Expression of an endochitinase gene from <Emphasis Type="Italic">Trichoderma virens</Emphasis> confers enhanced tolerance to <Emphasis Type="Italic">Alternaria</Emphasis> blight in transgenic <Emphasis Type="Italic">Brassica juncea</Emphasis> (L.) czern and coss lines

An endochitinase gene ‘ech42’ from the biocontrol fungus ‘Trichoderma virens’ was introduced to Brassica juncea (L). Czern and Coss via Agrobaterium tumefaciens mediated genetic transformation method. Integration and expression of the ‘ech42’ gene in transgenic lines were confirmed by PCR, RT-PCR and Southern hybridization. Transgenic lines (T₁) showed expected 3:1 Mendelian segregation ratio when segregation analysis for inheritance of transgene ‘hpt’ was carried out. Fluorimetric analysis of transgenic lines (T₀ and T₁) showed 7 fold higher endochitinase activity than the non-transformed plant. Fluorimetric zymogram showed presence of endochitinase (42 kDa) in crude protein extract of transgenic lines. In detached leaf bioassay with fungi Alternaria brassicae and Alternaria brassicicola, transgenic lines (T₀ and T₁) showed delayed onset of lesions as well as 30–73 % reduction in infected leaf area compared to non-transformed plant.     

Biological control of take-all in wheat by endophytic Bacillus subtilis E1R-j and potential mode of action

The bacterial strain E1R-j, isolated as an endophyte from wheat roots, exhibited high antifungal activity to Gaeumannomyces graminis var. tritici (Ggt). Strain E1R-j was identified as Bacillus subtilis based on morphological, physiological and biochemical methods as well as on 16S rDNA analysis. This strain inhibited mycelium growth in vitro of numerous plant pathogenic fungi, especially of Ggt, Coniothyrium diplodiella, Phomopsis sp. and Sclerotinia sclerotiorum. In greenhouse experiments, soil drenches with cell densities of 10⁶, 10⁹ and 10¹² CFU ml⁻¹ E1R-j reduced significantly take-all disease, caused by Ggt, in wheat seedling by 62.6%, 68.6% and 70.7%, respectively, compared to the inoculated control, 4 weeks after sowing. Growth parameters such as lengths and fresh weights of roots and shoots of Ggt-inoculated control plants were significantly lower compared to Ggt-inoculated and E1R-j treated plants. Field experiments in the season 2006/2007, heights of wheat plants in the Ggt inoculated plots were significantly reduced compared to the non inoculated treatments. Yield parameters such as kernels per head and thousand kernel weight (TKW) in inoculated control plants were lower compared to the other treatments. In the experimental year 2007/2008, independent treatments with the bacterial strain E1R-j and the fungicide Triadimefon reduced take-all disease in wheat roots by 55.3% and 61.9%, compared to the inoculated control plants. In this season plant height in inoculated control was significantly lower and also the yield parameters seeds per head and especially TKW were drastically reduced compared to the other treatments. E1R-j treatment alleviated the detrimental effects of take-all on grain yield parameters to a similar extent as Triadimefon application. SEM studies revealed that in the presence of E1R-j, hyphae of Ggt showed leakage, appeared ruptured, swollen and shriveled. Following root drench, strain E1R-j was able to colonize endophytically roots and leaves of wheat seedlings. While the population of the bacterial strain in wheat roots steadily increased from the second to the fourth leaf stage, in the leaf tissue the population of the strain rapidly declined. TEM studies also showed that cells of E1R-j were present in roots of wheat seedlings and effectively retarded infection and colonization of Ggt in root tissue; suppression of Ggt by E1R-j was accompanied by disintegration of hyphal cytoplasm. In addition, in the presence of E1R-j cells in Ggt-infected root tissue morphological defense reactions were triggered such as formation of wall appositions and papillae. The results presented indicate that the endophytic strain E1R-j of B. subtilis meets demands required for biocontrol of take-all.     

Cloning of TPS gene from eelgrass species Zostera marina and its functional identification by genetic transformation in rice

The full-length cDNA sequence (2613 bp) of the trehalose-6-phosphate synthase (TPS) gene of eelgrass Zostera marina (ZmTPS) was identified and cloned. Z. marina is a kind of seed-plant growing in sea water during its whole life history. The open reading frame (ORF) region of ZmTPS gene encodes a protein of 870 amino acid residues and a stop codon. The corresponding genomic DNA sequence is 3770 bp in length, which contains 3 exons and 2 introns. The ZmTPS gene was transformed into rice variety ZH11 via Agrobacterium-mediated transformation method. After antibiotic screening, molecular characterization, salt-tolerance and trehalose content determinations, two transgenic lines resistant to 150 mM NaCL solutions were screened. Our study results indicated that the ZmTPS gene was integrated into the genomic DNA of the two transgenic rice lines and could be expressed well. Moreover, the detection of the transformed ZmTPS gene in the progenies of the two transgenic lines was performed from T₁ to T₄ generations; and results suggested that the transformed ZmTPS gene can be transmitted from parent to the progeny in transgenic rice.     

The expression of a maize glutathione S-transferase gene in transgenic wheat confers herbicide tolerance,both in planta and in vitro

Maize (Zea mays), in common with a number of other important crop species, has several glutathione S-transferase (GST) isoforms that have been implicated in the detoxification of xenobiotics via glutathione conjugation. A cDNA encoding the maize GST subunit GST-27, under the control of a strong constitutive promoter, was introduced into explants of the wheat (Triticum aestivum L.) lines cv. Florida and L88-31 via particle bombardment, using the phosphinothricin acetyltransferase (pat) gene as a selectable marker. All six independent transgenic wheat lines recovered expressed the GST-27 gene. T₁ progeny of these wheat lines were germinated on solid medium containing the chloroacetanilide herbicide alachlor, and tolerance to this herbicide was correlated with GST-27 expression levels. In glasshouse sprays, homozygous T₂ plants were resistant not only to alachlor but also to the chloroacetanilide herbicide dimethenamid and the thiocarbamate herbicide EPTC. These additional GST-27 activities, demonstrated via over-expression in a heterologous host, have not been described previously. T₂ plants showed no enhanced tolerance to the herbicides atrazine (an s-triazine) or oxyfluorfen (a diphenyl ether). In further experiments, T₂ wheat plants were recovered from immature transgenic scutella cultured on medium containing 100 mg/l alachlor, a concentration which killed null segregant and wild-type scutella. These data indicate the potential of the maize GST-27 gene as a selectable marker in wheat transformation.     

Stably expressed <Emphasis Type="SmallCaps">d</Emphasis>-genome-derived HMW glutenin subunit genes transformed into different durum wheat genotypes change dough mixing properties

Durum wheat (Triticum turgidum L. var. durum) is traditionally used for the production of numerous types of pasta, and significant amounts are also used for bread-making, particularly in southern Italy. The research reported here centres on the glutenin subunits 1Dx5 and 1Dy10 encoded by chromosome 1D, and whose presence in hexaploid wheats is positively correlated with higher dough strength. In order to study the effects of stable expression of the 1Dx5 and 1Dy10 glutenin subunits in different durum wheat genotypes, four cultivars commonly grown in the Mediterranean area (‘Svevo’, ‘Creso’, ‘Varano’ and ‘Latino’) were co-transformed, via particle bombardment of cultured immature embryos, with the two wheat genes Glu-D1-1d and Glu-D1-2b encoding the glutenin subunits, and a third plasmid containing the bar gene as a selectable marker. Protein gel analyses of T₁ generation seed extracts showed expression of one or both glutenin genes in four different transformed durum wheat plants. One of these transgenic lines, DC2-65, showed co-suppression of all HMW-GS, including the endogenous ones. Transgene stability in the transgenic lines has been studied over four generations (T₁–T₄). Fluorescence in situ hybridization (FISH) analysis of metaphase chromosomes from T₄ plants showed that the integration of transgenes occurred in both telomeric and centromeric regions. The three plasmids were found inserted at a single locus in two lines and in two loci on the same chromosome arm in one line. The fourth line had two transgenic loci on different chromosomes: one with both glutenin plasmids and a different one containing only the construct with the gene encoding the 1Dy10 glutenin subunit. Segregation of these two loci in subsequent generations allowed establishment of two sublines, one containing both 1Dx5 and 1Dy10 and the other containing only 1Dy10. Small-scale quality tests showed that accumulation of Dx5, Dy10 or both in transgenic durum wheat seeds resulted in doughs with stronger mixing characteristics. A. Gadaleta and A. E. Blechl have contributed equally to this work.