Recent development of vectors and methodologies to introduce recombinant DNA into members of the genus Mycobacterium has provided new approaches for investigating these important bacteria. While most pathogenic mycobacteria are slow-growing, Mycobacterium smegmatis is a fast-growing, non-pathogenic species that has been used for many years as a host for mycobacteriophage propagation and, recently, as a host for the introduction of recombinant DNA. Its use as a cloning host for the analysis of mycobacterial genes has been limited by its inability to be efficiently transformed with plasmid vectors. This work describes the isolation and characterization of mutants of M. smegmatis that can be transformed, using electroporation, at efficiencies 10(4) to 10(5) times greater than those of the parent strain, yielding more than 10(5) transformants per microgram of plasmid DNA. The mutations conferring this efficient plasmid transformation (Ept) phenotype do not affect phage transfection or the integration of DNA into the M. smegmatis chromosome, but seem to be specific for plasmid transformation. Such Ept mutants have been used to characterize plasmid DNA sequences essential for replication of the Mycobacterium fortuitum plasmid pAL5000 in mycobacteria by permitting the transformation of a library of hybrid plasmid constructs. Efficient plasmid transformation of M. smegmatis will facilitate the analysis of mycobacterial gene function, expression and replication and thus aid in the development of BCG as a multivalent recombinant vaccine vector and in the genetic analysis of the virulence determinants of pathogenic mycobacteria.

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The evolution of the ability to synthesize specialized metabolites is likely to have been key for survival and diversification of different plant species. Oats (Avena spp.) produce antimicrobial triterpenoids (avenacins) that protect against disease. The oat ␤-amyrin synthase gene AsbAS1, which encodes the first committed enzyme in the avenacin biosynthetic pathway, is clearly distinct from other plant ␤-amyrin synthases. Here we show that AsbAS1 has arisen by duplication and divergence of a cycloartenol synthase-like gene, and that its properties have been refined since the divergence of oats and wheat. Strikingly, we have also found that AsbAS1 is clustered with other genes required for distinct steps in avenacin biosynthesis in a region of the genome that is not conserved in other cereals. Because the components of this gene cluster are required for at least four clearly distinct enzymatic processes (2,3-oxidosqualene cyclization, ␤-amyrin oxidation, glycosylation, and acylation), it is unlikely that the cluster has arisen as a consequence of duplication of a common ancestor.

CYP51 sterol demethylases are the only cytochrome P450 enzymes with a conserved function across the animal, fungal, and plant kingdoms (in the synthesis of essential sterols). These highly conserved enzymes, which are important targets for cholesterollowering drugs, antifungal agents, and herbicides, are regarded as the most ancient member cytochrome P450 family. Here we present a report of a CYP51 enzyme that has acquired a different function. We show that the plant enzyme AsCYP51H10 is dispensable for synthesis of essential sterols and has been recruited for the production of antimicrobial compounds (avenacins) that confer disease resistance in oats. The AsCyp51H10 gene is synonymous with Sad2, a gene that we previously had defined by mutation as being required for avenacin synthesis. In earlier work, we showed that Sad1, the gene encoding the first committed enzyme in the avenacin pathway (␤-amyrin synthase), had arisen by duplication and divergence of a cycloartenol synthase-like gene. Together these data indicate an intimate evolutionary connection between the sterol and avenacin pathways. Sad1 and Sad2 lie within 70 kb of each other and are expressed specifically in the epidermal cells of the root tip, the site of accumulation of avenacins. These findings raise intriguing questions about the recruitment, coevolution, and regulation of the components of this specialized defense-related metabolic pathway.Avena ͉ disease resistance ͉ oat ͉ metabolic diversity ͉ gene duplication

To meet the challenge of feeding a growing population, breeders and scientists are continuously looking for ways to increase genetic gain in crop breeding. One way this can be achieved is through 'speed breeding' (SB), which shortens the breeding cycle and accelerates research studies through rapid generation advancement. The SB method can be carried out in a number of ways, one of which involves extending the duration of a plant's daily exposure to light (photoperiod) combined with early seed harvest in order to cycle quickly from seed to seed, thereby reducing the generation times for some long-day (LD) or day-neutral crops. Here we present glasshouse and growth chamber-based SB protocols with supporting data from experimentation with several crop species. These protocols describe the growing conditions, including soil media composition, lighting, temperature and spacing, which promote rapid growth of spring and winter bread wheat, durum wheat, barley, oat, various members of the Brassica family, chickpea, pea, grasspea, quinoa and the model grass Brachypodium distachyon. Points of flexibility within the protocols are highlighted, including how plant density can be increased to efficiently scale-up plant numbers for single seed descent (SSD) purposes. Conversely, instructions on how to perform SB on a small-scale by creating a benchtop SB growth cabinet that enables optimization of parameters at a low cost are provided. We also outline the procedure for harvesting and germinating premature wheat, barley and pea seed to reduce generation time. Finally, we provide troubleshooting suggestions to avoid potential pitfalls.

Here we show the novel oxidosqualene cyclase AsbAS1 catalyzes the first committed step in the synthesis of antifungal triterpenoid saponins that accumulate in oat roots. We also demonstrate that two sodium azide-generated saponin-deficient mutants of oat, which define the Sad1 genetic complementation group, are defective in the gene encoding this enzyme and provide molecular genetic evidence indicating a direct link between AsbAS1, triterpenoid saponin biosynthesis, and disease resistance. Orthologs of AsbAS1 are absent from modern cereals and may have been lost during selection, raising the possibility that this gene could be exploited to enhance disease resistance in crop plants.

authors request that the following clarification be noted. The Methods and Materials on p. 6987, line 28, indicated that mycobacterial cultures were grown in Middlebrook 7H9 broth (Difco) supplemented with ADC (albumin-dextrose complex) and 0.05% Tween 80. Middlebrook broth as originally formulated (23) and subsequently used for mycobacteria (24) contains glycerol. It has been brought to the authors' attention that the label on the Middlebrook-Dubos medium currently supplied by Difco states "if desired [each liter may contain] 2 ml glycerol or 0.05% Tween" (italics added). For the purposes of genetic transformation of BCG, the authors find that inclusion of 0.2% glycerol, as in the standard formulation, is required.

Serine carboxypeptidase-like (SCPL) proteins have recently emerged as a new group of plant acyltransferases. These enzymes share homology with peptidases but lack protease activity and instead are able to acylate natural products. Several SCPL acyltransferases have been characterized to date from dicots, including an enzyme required for the synthesis of glucose polyesters that may contribute to insect resistance in wild tomato (Solanum pennellii) and enzymes required for the synthesis of sinapate esters associated with UV protection in Arabidopsis thaliana. In our earlier genetic analysis, we identified the Saponin-deficient 7 (Sad7) locus as being required for the synthesis of antimicrobial triterpene glycosides (avenacins) and for broad-spectrum disease resistance in diploid oat (Avena strigosa). Here, we report on the cloning of Sad7 and show that this gene encodes a functional SCPL acyltransferase, SCPL1, that is able to catalyze the synthesis of both N-methyl anthraniloyl-and benzoyl-derivatized forms of avenacin. Sad7 forms part of an operon-like gene cluster for avenacin synthesis. Oat SCPL1 (SAD7) is the founder member of a subfamily of monocot-specific SCPL proteins that includes predicted proteins from rice (Oryza sativa) and other grasses with potential roles in secondary metabolism and plant defense.