T4 Lysozyme and Attacin Genes Enhance Resistance of Transgenic `Galaxy' Apple against Erwinia amylovora

Abstract

<jats:p>Genes encoding lysozyme (<jats:italic>T4L</jats:italic>) from T4 bacteriophage and attacin E (<jats:italic>att</jats:italic>E) from <jats:italic>Hyalophora cecropia</jats:italic> were used, either singly or in combination, to construct plant binary vectors, pLDB15, p35SAMVT4, and pPin2Att35SAMVT4, respectively, for <jats:italic>Agrobacterium</jats:italic>-mediated transformation of `Galaxy' apple, to enhance resistance to <jats:italic>Erwinia amylovora</jats:italic>. In these plasmids, the <jats:italic>T4L</jats:italic> gene was controlled by the cauliflower mosaic virus 35S promoter with duplicated upstream domain and the untranslated leader sequence of alfalfa mosaic virus RNA 4, and the <jats:italic>att</jats:italic>E gene was controlled by the potato proteinase inhibitor II (<jats:italic>Pin2</jats:italic>) promoter. All transgenic lines were screened by polymerase chain reaction (PCR) for <jats:italic>T4L</jats:italic> and <jats:italic>att</jats:italic>E genes, and a double-antibody sandwich enzyme-linked immunosorbent assay for neomycin phosphotransferase II. Amplification of <jats:italic>T4L</jats:italic> and <jats:italic>att</jats:italic>E genes was observed in reverse transcriptase-PCR, indicating that these genes were transcribed in all tested transgenic lines containing each gene. The attacin protein was detected in all <jats:italic>att</jats:italic>E transgenic lines. The expression of <jats:italic>att</jats:italic>E under the <jats:italic>Pin2</jats:italic> promoter was constitutive but higher levels of expression were observed after mechanical wounding. Some <jats:italic>T4L</jats:italic> or <jats:italic>att</jats:italic>E transgenic lines had significant disease reduction compared to nontransgenic `Galaxy'. However, transgenic lines containing both <jats:italic>att</jats:italic>E and <jats:italic>T4L</jats:italic> genes were not significantly more resistant than nontransgenic `Galaxy', indicating that there was no in planta synergy between <jats:italic>att</jats:italic>E and <jats:italic>T4L</jats:italic> with respect to resistance to <jats:italic>E</jats:italic>. <jats:italic>amylovora</jats:italic>.</jats:p>

Ectopic expression of a novel peach (Prunus persica) CBF transcription factor in apple (Malus × domestica) results in short-day induced dormancy and increased cold hardiness

“…Explants were propagated in tissue culture as described by Norelli et al (1988) and Ko et al (2002), with root induction as described by Bolar et al (1998). Young trees were transferred to soil and grown in a greenhouse with Na vapor supplemental lighting to maintain the daylength at 16 h, and a maximum-minimum temperature range of 35/20°C.…”

Transformation of apple ( Malus domestica Borkh.) with the stilbene synthase gene from grapevine ( Vitis vinifera L.) and a PGIP gene from kiwi ( Actinidia deliciosa )

“…With the target of increasing disease resistance to fungal pathogens, Bolar et al (2000Bolar et al ( , 2001 and Wong et al (1999) introduced a chitinase from Trichoderma harzianum into Marshall McIntosh and Royal Gala. The lytic proteins attacin and T4 lysozyme (Norelli et al 1994;Ko et al 2000Ko et al , 2002 and a modified cecropin MB39 gene (Liu et al 2001) were used to achieve enhanced resistance to fire blight, caused by the bacterium Erwinia amylovora.…”

Transgenic Sweet Orange (Citrus sinensis L. Osbeck) Expressing the attacin A Gene for Resistance to Xanthomonas citri subsp. citri

“…Attacin, cecropin, and sarcotoxin genes, derived from different insect genomes, have been introduced in different plant species with promising results (Reynoird et al 1999;Ko et al 2000Ko et al , 2002Bespalhok Filho et al 2001). Transgenic potato (Solanum tuberosum L.) expressing cecropin and tobacco (Nicotiana tabacum L.) expressing shiva-1 genes (a cecropin synthetic analog) showed an increase in resistance to Ralstonia solanacearum Smith Yabuuchi et al (Montanelli and Nascari 1991;Jaynes et al 1993).…”

“…Mansf.] expressing the basic attA form (Norelli and Aldwinckle 1993) as well as transgenic apple and pear (Pyrus communis L.) expressing the acidic attE form (Norelli et al 1994;Ko et al 2002;Reynoird et al 1999) showed a reduced susceptibility to fire blight Erwinia amylovora (Burr) Winslow et al…”