Transgenic apple plants overexpressing the Lc gene of maize show an altered growth habit and increased resistance to apple scab and fire blight

Flachowsky, Henryk; Szankowski, I.; Fischer, Thilo C.; Richter, Klaus; Peil, Andreas; Höfer, Monika; Dörschel, Claudia; Schmoock, Sylvia; Gau, Achim E.; Halbwirth, Heidi; Hanke, Magda‐Viola

doi:10.1007/s00425-009-1074-4

Cited by 45 publications

(21 citation statements)

References 49 publications

(65 reference statements)

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“…Recently, constitutive expression in cv Holsteiner Cox of the maize (Zea mays) regulatory leaf color (Lc) gene, which encodes a transcription factor, resulted in both increased transcript levels of flavonoid biosynthetic enzymes and increased concentrations of specific flavonoid classes, such as anthocyanins, flavan-3-ols, and proanthocyanidins (Li et al, 2007). These Lc-transgenic apple plants exhibited improved resistance to fire blight and scab (Flachowsky et al, 2010). Immunofluorescence localization of BIS3 in the transition zone of fire-blight-infected stems of cv Holsteiner Cox revealed the presence of the enzyme protein in the parenchyma of the bark.…”

Section: Discussionmentioning

confidence: 99%

Differential Expression of Biphenyl Synthase Gene Family Members in Fire-Blight-Infected Apple ‘Holsteiner Cox’

Chizzali

Gaid²,

Belkheir³

et al. 2011

Plant Physiol.

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Fire blight, caused by the bacterium Erwinia amylovora, is a devastating disease of apple (Malus 3 domestica). The phytoalexins of apple are biphenyls and dibenzofurans, whose carbon skeleton is formed by biphenyl synthase (BIS), a type III polyketide synthase. In the recently published genome sequence of apple 'Golden Delicious', nine BIS genes and four BIS gene fragments were detected. The nine genes fall into four subfamilies, referred to as MdBIS1 to MdBIS4. In a phylogenetic tree, the BIS amino acid sequences from apple and Sorbus aucuparia formed an individual cluster within the clade of the functionally diverse type III polyketide synthases. cDNAs encoding MdBIS1 to MdBIS4 were cloned from fire-blight-infected shoots of apple 'Holsteiner Cox,' heterologously expressed in Escherichia coli, and functionally analyzed. Benzoyl-coenzyme A and salicoylcoenzyme A were the preferred starter substrates. In response to inoculation with E. amylovora, the BIS3 gene was expressed in stems of cv Holsteiner Cox, with highest transcript levels in the transition zone between necrotic and healthy tissues. The transition zone was the accumulation site of biphenyl and dibenzofuran phytoalexins. Leaves contained transcripts for BIS2 but failed to form immunodetectable amounts of BIS protein. In cell cultures of apple 'Cox Orange,' expression of the BIS1 to BIS3 genes was observed after the addition of an autoclaved E. amylovora suspension. Using immunofluorescence localization under a confocal laser-scanning microscope, the BIS3 protein in the transition zone of stems was detected in the parenchyma of the bark. Dot-shaped immunofluorescence was confined to the junctions between neighboring cortical parenchyma cells.

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Section: Discussionmentioning

confidence: 99%

Differential Expression of Biphenyl Synthase Gene Family Members in Fire-Blight-Infected Apple ‘Holsteiner Cox’

Chizzali

Gaid²,

Belkheir³

et al. 2011

Plant Physiol.

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“…Transformation for developing resistance to EAB in pumpkin ash will be a feasible approach because of transformation attainability in both model and non-model woody species, as well as closely related green ash (Du and Pijut 2009;Flachowsky et al 2010;Noel et al 2005). To date there has been no research reported on establishing pumpkin ash in tissue culture.…”

Section: Introductionmentioning

confidence: 98%

Hypocotyl derived in vitro regeneration of pumpkin ash (Fraxinus profunda)

Stevens

Pijut

2011

Plant Cell Tiss Organ Cult

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Pumpkin ash (Fraxinus profunda (Bush) Bush) is at risk for extirpation by an exotic insect, the emerald ash borer (EAB). Pumpkin ash is limited to wetland areas of the Eastern United States, and has been listed as an endangered species because of EAB activity. Pumpkin ash provides many benefits to the ecosystem, and its wood is used in the manufacturing industry. In vitro regeneration provides an integral tool for the mass propagation and genetic transformation of pumpkin ash to combat EAB. Therefore, a plant regeneration protocol was developed for pumpkin ash. Aseptically extracted hypocotyls formed adventitious shoots following 4 weeks on Murashige and Skoog (MS) medium supplemented with 0-22.2 lM 6-benzyladenine (BA) and 0-6.8 lM thidiazuron (TDZ) then transferred for an additional 4 weeks on MS medium with Gamborg B5 vitamins plus 0.2 g L -1 glycine (B5G) containing 6.7 lM BA, 1 lM indole-3-butryic acid (IBA), and 0.29 lM gibberellic acid (GA 3 ). As adventitious shoots developed, these were transferred to a MSB5G medium with 13.3 lM BA, 1 lM IBA, and 0.29 lM GA 3 for shoot elongation. Elongated shoots were successfully micropropagated using MSB5 medium with 10 lM BA and 10 lM TDZ. Adventitious root formation was as high as 94% using woody plant medium supplemented with 4.9 lM IBA with shoots cultured for 10 days in the dark followed by culture under a 16-h photoperiod.Acclimatization to the greenhouse was successful and normal plant growth was observed. This protocol will provide a means for genetic transformation for EAB resistance and mass propagation for conservation.

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“…Finally, 5 lL of the PCR product were run in 1.5% agarose gel colored by Sybrgreen (Sigma-Aldrich) and visualized under UV light. Seven reference genes such as GAPDH [3], 18S rRNA [3], S19 [3], Ubiquitine [2], Rubisco [4], RNA polymerase [4] and b-actine [9] as well as for the two viruses ASGV and ApMV [12] were chosen. These internal genes are known as the most stable expression in apple tissues Table 1.…”

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Evaluation of Reference Genes for the Relative Quantification of Apple stem grooving virus and Apple mosaic virus in Apple Trees

Gadiou

Kundu

2012

Indian J. Virol.

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A SYBR Green Ò -based one step RT-qPCR assay was developed for the detection and quantification of Apple stem grooving virus (ASGV) and Apple mosaic virus (ApMV). The RT-qPCR assay employed seven plantexpressed genes-glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA, ubiquitin, ribosomal protein S19, Rubisco, RNA polymerase subunit II and bactin-as internal reference housekeeping genes in a relative quantification system in three apple cultivars (i.e. Idared, Champion, Fragrance). The average expression stability (M) found by GeNorm software suggest that GAPDH and S19 were the most stable reference genes. We propose employing GAPDH and S19 as housekeeping genes for accurate quantification of ASGV and ApMV in apple leaf samples. The detection limit for both viruses was found around 70 copies of viral genome by one-step RT-qPCR.The fluorescence-based quantitative real-time PCR (qPCR) has been used to detect and measure nucleic acids of a wide range of plant viruses with RNA and DNA genome [13,16,17]. Two main qPCR based techniques: relative and absolute quantification have emerged from various studies on virus detection and quantification of virus titer. The ''absolute quantification'' with known standard is the most commonly used for plant virus quantification. On the other hand, relative quantification uses a comparative quantification with internal reference endogenous housekeeping control genes [1]. The relative quantification determines the changes in RNA levels of a gene across multiple samples and expresses it relative to the levels of an internal control RNA. Therefore, relative quantification does not require standards with known concentrations [15]. Relative quantification employing reference internal control genes was used for several plant virus detection and quantification studies [5,6]. A comparative study showed that the relative quantification assay may be as accurate as absolute quantification when appropriate housekeeping genes are used and normalized with target genes [6,10]. In this paper we have chosen endogenous housekeeping genes for quantification of viruses in apple trees in a relative quantification system. Two apple tree infecting viruses, Apple stem grooving virus (ASGV) and Apple mosaic virus (ApMV) are selected. ASGV and ApMV belong to the most frequently emerged pathogens encounters in pome fruit trees. Several molecular-based methods have been elaborated to perform routine detection [7,8,11,12]. However, a highly sensitive real-time based detection and quantification assay for these viruses is missing so far. We described here a SYBER green based relative quantification assay employing appropriate endogenous reference genes as internal control.A total of 39 samples coming from different apple cultivars grown in vitro culture (i.e. Idared, Champion, Fragrance) healthy (14 plants) and infected (25 plants) have been used to validate the reference genes as well as the target genes. The leaves of plant material were grinded in liquid nitrogen and the total RNA ...

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Transgenic apple plants overexpressing the Lc gene of maize show an altered growth habit and increased resistance to apple scab and fire blight

Cited by 45 publications

References 49 publications

Differential Expression of Biphenyl Synthase Gene Family Members in Fire-Blight-Infected Apple ‘Holsteiner Cox’

Differential Expression of Biphenyl Synthase Gene Family Members in Fire-Blight-Infected Apple ‘Holsteiner Cox’

Hypocotyl derived in vitro regeneration of pumpkin ash (Fraxinus profunda)

Evaluation of Reference Genes for the Relative Quantification of Apple stem grooving virus and Apple mosaic virus in Apple Trees

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