Bacterial endophytes from wild and ancient maize are able to suppress the fungal pathogen Sclerotinia homoeocarpa.

AIMS: The aim of this study was to determine if endophytes from wild and ancient Zea plants (corn family) have anti-fungal activities, specifically against the most important fungal pathogen (Sclerotinia homoeocarpa) of creeping bentgrass, a relative of Zea, used here as a model grass. METHODS AND RESULTS: A library of 190 bacterial endophytes from wild, ancient and modern Zea plants were tested for their ability to suppress S. homoeocarpa in vitro, followed by in planta testing of candidates using greenhouse trials. Three endophytes could suppress S. homoeocarpa, originating from wild maize and an ancient Mexican landrace, consistent with our hypothesis. 16S phylogenetic analysis and BOX-PCR DNA fingerprinting suggest that the anti-fungal endophytes are distinct strains of Burkholderia gladioli. One strain (3A12) was confirmed to colonize creeping bentgrass using green fluorescent protein (GFP) tagging. Evans blue vitality staining demonstrated that the bacterial endophytes exhibited fungicidal activities against the pathogen. The endophytes inhibited a wide spectrum of plant-associated fungi including diverse crop pathogens. CONCLUSIONS: The results support the hypothesis that wild and ancient Zea genotypes host bacterial endophytes that can control fungal pathogen(s). SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that wild and ancient crops may be an unexplored reservoir of anti-fungal bacterial endophytes.

Somatic and germinal mobility of the RescueMu transposon in transgenic maize.

RescueMu, a Mu1 element containing a bacterial plasmid, is mobilized by MuDR in transgenic maize. Somatic excision from a cell-autonomous marker gene yields >90% single cell sectors; empty donor sites often have deletions and insertions, including up to 210 bp of RescueMu/Mu1 terminal DNA. Late somatic insertions are contemporaneous with excisions, suggesting that "cut-and-paste" transposition occurs in the soma. During reproduction, RescueMu transposes infrequently from the initial transgene array, but once transposed, RescueMu is suitable for high throughput gene mutation and cloning. As with MuDR/Mu elements, heritable RescueMu insertions are not associated with excisions. Both somatic and germinal RescueMu insertions occur preferentially into genes and gene-like sequences, but they exhibit weak target site preferences. New insights into Mu behaviors are discussed with reference to two models proposed to explain the alternative outcomes of somatic and germinal events: a switch from somatic cut-and-paste to germinal replicative transposition or to host-mediated gap repair from sister chromatids.

A maize MuDR transposon promoter shows limited autoregulation.

Transgenic maize expressing luciferase under the control of the mudrB terminal inverted repeat promoter (TIRB) of the MuDR transposon was assayed for transgene expression in active and inactive Mutator lines. We find that active MuDR elements increase TIRB-luciferase expression by 2- to 10-fold, relative to nonMuDR or silenced MuDR lines, in embryonic leaves in 75% of plants tested. However, this increase does not persist in juvenile and adult leaves. In pollen, TIRB-luciferase expression is up to 100-fold higher than in leaves but is unaffected by the presence or absence of active MuDR. Because the MuRA transposase binds to a motif within TIRB, we hypothesize that MURA may act as a weak transcriptional activator of TIRB or may partly inhibit host-induced silencing of TIRB in active Mutator lines during the early stages of somatic growth. Our results contrast with those for the maize transposon Spm, in which the TNPA transposase acts as a repressor of the Spm promoter in active Spm lines.

The MuDR transposon terminal inverted repeat contains a complex plant promoter directing distinct somatic and germinal programs.

The Mu transposons of maize are under stringent developmental control. Elements excise at high frequencies in terminally dividing somatic cells, but not in meristems. Mu elements in germinal cells amplify, without excision, and insert throughout the genome. All activities require MuDR, which encodes two genes, mudrA and mudrB, whose near-identical promoters are located in the transposon terminal inverted repeats (TIR). We have fused the 216 bp TIR of the mudrB gene to GUS and luciferase reporters. We demonstrate that TIRB programs reporter expression in diverse, meristematic somatic cells, paradoxically in those cells in which Mu excisions are repressed. In germinal cells, immature tassel and mature pollen, reporter expression increases up to 20-fold compared to leaf. By RNA blot hybridization, we demonstrate that endogenous mudrB and mudrA transcripts increase significantly in mature pollen; sequence comparisons demonstrate that the MuDR TIRs contain plant cell-cycle enhancer motifs and functionally defined pollen enhancers. Therefore, the MuDR TIR promoters are developmentally regulated in both somatic and germinal tissues. Because database sequence analysis suggests that the MuDR TIR enhancers should be functional in both monocots and dicots, we suggest that the native MuDR promoter be used in attempts to transfer the unique behavior of Mu transposition to heterologous hosts.

The late developmental pattern of Mu transposon excision is conferred by a cauliflower mosaic virus 35S -driven MURA cDNA in transgenic maize.

The MuDR element responsible for Mutator activities in maize encodes two genes, mudrA and mudrB. Each encodes multiple transcripts hypothesized to regulate, directly or indirectly, the unique late timing and switch in transposition mechanism during maize development. mudrA, which encodes the MURA transposase, is unstable in bacterial plasmids, a technical problem solved by using phage M13 as a vector to prepare DNA for biolistic transformation. In transgenic maize, a single 2.7-kb mudrA cDNA predicted to encode an 823-amino acid protein is sufficient to catalyze late somatic excisions, despite removal of the native promoter, alternative transcription start sites, known introns, polymorphic 5' and 3' untranslated sequences, and the mudrB gene. These results suggest that post-translational regulation confers Mu excision timing. The transgene is active in lines containing silencing MuDR elements. This suggests that endogenous MuDR transposons do not measurably immunize the host against expression of a homologous transgene.