Agrobacterium is widely considered to be the only bacterial genus capable of transferring genes to plants. When suitably modified, Agrobacterium has become the most effective vector for gene transfer in plant biotechnology. However, the complexity of the patent landscape has created both real and perceived obstacles to the effective use of this technology for agricultural improvements by many public and private organizations worldwide. Here we show that several species of bacteria outside the Agrobacterium genus can be modified to mediate gene transfer to a number of diverse plants. These plant-associated symbiotic bacteria were made competent for gene transfer by acquisition of both a disarmed Ti plasmid and a suitable binary vector. This alternative to Agrobacterium-mediated technology for crop improvement, in addition to affording a versatile 'open source' platform for plant biotechnology, may lead to new uses of natural bacteria-plant interactions to achieve plant transformation.

The substrate-specific induction of wheat (Trificum aestivum 1. cv Fenman) leaf cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) was examined in relation to its role in regulating the composition of defensive lignin induced at wound margins. Treatment of wounds with a partially acetylated chitosan hydrolysate or spores of the nonpathogen Bofryfis cinerea elicited lignification at wound margins and invoked significant increases in phenylalanine ammonia-lyase (EC 4.3.1.5), peroxidase (EC 1.11.1.7), and CAD activities. The substrate-specific induction of CAD with time was determined in elicitor-treated leaves and in excised lignifying wounds. In whole leaf extracts no significant increases in p-coumaryl and coniferyl alcohol dehydrogenase adivities were detedable, but a significant 5-fold increase in sinapyl alcohol dehydrogenase adivity was evident 32 h after elicitor treatment. Similarly, fungal challenge resulted in elevated levels of only sinapyl alcohol dehydrogenase in whole-leaf extrads. In excised lignifying tissues p-coumaryl alcohol dehydrogenase levels were similar to those observed in healthy tissue. A small yet significant increase in coniferyl alcohol dehydrogenase was apparent, but the most dramatic increase occurred in sinapyl alcohol dehydrogenase adivity, which increased to values approximately 10 times higher than the untreated controls. Our results show for the first time that CAD induction in lignifying tissués of wheat is predominantly attributable to highly localized increases in sinapyl alcohol dehydrogenase adivity.

Expression of the fis1 gene from flax (Linum usitatissimum) is induced by a compatible rust (Melampsora lini) infection. Infection of transgenic plants containing a ␤-glucuronidase (GUS) reporter gene under the control of the fis1 promoter showed that induction is highly localized to those leaf mesophyll cells within and immediately surrounding rust infection sites. The level of induction reflects the extent of fungal growth. In a strong resistance reaction, such as the hypersensitive fleck mediated by the L6 resistance gene, there is very little fungal growth and a microscopic level of GUS expression. Partially resistant flax leaves show levels of GUS expression that were intermediate to the level observed in the fully susceptible infection. Sequence and deletion analysis using both transient Agrobacterium tumefaciens expression and stable transformation assays have shown that the rust-inducible fis1 promoter is contained within a 580-bp fragment. Homologs of fis1 were identified in expressed sequence tag databases of a range of plant species including dicots, monocots, and a gymnosperm. Homologous genes isolated from maize (Zea mays; mis1), barley (Hordeum vulgare; bis1), wheat (Triticum aestivum; wis1), and Arabidopsis encode proteins that are highly similar (76%-82%) to the FIS1 protein. The Arabidopsis homologue has been reported to encode a ⌬ 1 -pyrroline-5-carboxylate dehydrogenase that is involved in the catabolism of proline to glutamate. RNA-blot analysis showed that mis1 in maize and the bis1 homolog in barley are both up-regulated by a compatible infection with the corresponding species-specific rust. The rust-induced genes homologous to fis1 are present in many plants. The promoters of these genes have potential roles for the engineering of synthetic rust resistance genes by targeting transgene expression to the sites of rust infection.For successful parasitism of plants by fungal pathogens, particularly in the case of obligate, biotrophic phytopathogens such as rust fungi, it is apparent that the pathogen has evolved strategies for manipulating host cellular metabolism, morphology, and development (Clancy and Coffey, 1980; Sutton and Shaw, 1982, 1986; Scholes, 1992; Roy, 1993; Chou et al., 2000; Hall and Williams, 2000). Successful infection of the host plant by the pathogen requires the induction of a subset of pathogen genes that are essential for pathogenicity (Pieterse et al., 1993; Talbot et al., 1993; Rogers et al., 1994; Hwang et al., 1995; Hahn and Mengden, 1997; Hahn et al., 1997). Less clear is the role and expression of specific host genes that may be required for successful pathogen infection of the plant. Identification of host genes induced during a successful pathogen infection may provide insight into the compatible host/pathogen interaction at a molecular level.The fis1 gene of flax (Linum usitatissimum) was identified as a host gene up-regulated during a successful or compatible flax rust (Melampsora lini) infection (Roberts and Pryor, 1995). RNA-blot analysis of fis1 demonstr...

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The induction and substrate speci®city of cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) was investigated in relation to the deposition of a defensive, syringyl-rich lignin at wound margins in wheat (Triticum aestivum L. cv. Brigadier). Column chromatography of untreated, wounded and elicitor-treated tissues revealed three major CAD forms (CAD-A, -B and -C) of which only CAD-C was responsive to elicitors. Examination of the substrate preference of these fractions indicated p-coumaryl alcohol to be the preferred substrate of CAD-A and CAD-B, whereas sinapyl alcohol was favoured by CAD-C. Activity-stained isoelectric focussing gels revealed in untreated and wounded leaves four CAD isoenzymes with isoelectric points of 4.59 (i), 4.67 (ii), 4.81 (iii), 4.93 (iv). Elicitor treatment generally enhanced the staining of all isoenzymes and resulted in the appearance of two new isoenzymes of 5.22 (v) and pI 5.31 (vi). In activity stained non-denaturing PAGE gels, CAD-C further resolved into two distinct zones of CAD activity. Cinnamyl alcohol dehydrogenase-C was puri®ed to apparent homogeneity and characterisation revealed a 45-kDa subunit peptide which in its native form demonstrated a marked substrate preference for sinapyl alcohol. Overall, the dierential induction and substrate preference of CAD-C are consistent with a defensive role during defensive ligni®cation at wound margins in wheat.Abbreviations: CAD = cinnamyl alcohol dehydrogenase; IEF = isoelectric focussing; pI = isoelectric point Correspondence to: M.S. Barber;