Jon Duvick

Conserved Subgroups and Developmental Regulation in the Monocot rop Gene Family

Rop small GTPases are plant-specific signaling proteins with roles in pollen and vegetative cell growth, abscisic acid signal transduction, stress responses, and pathogen resistance. We have characterized the rop family in the monocots maize (Zea mays) and rice (Oryza sativa). The maize genome contains at least nine expressed rops, and the fully sequenced rice genome has seven. Based on phylogenetic analyses of all available Rops, the family can be subdivided into four groups that predate the divergence of monocots and dicots; at least three have been maintained in both lineages. However, the Rop family has evolved differently in the two lineages, with each exhibiting apparent expansion in different groups. These analyses, together with genetic mapping and identification of conserved non-coding sequences, predict orthology for specific rice and maize rops. We also identified consensus protein sequence elements specific to each Rop group. A survey of ROP-mRNA expression in maize, based on multiplex reverse transcriptase-polymerase chain reaction and a massively parallel signature sequencing database, showed significant spatial and temporal overlap of the nine transcripts, with high levels of all nine in tissues in which cells are actively dividing and expanding. However, only a subset of rops was highly expressed in mature leaves and pollen. Intriguingly, the grouping of maize rops based on hierarchical clustering of expression profiles was remarkably similar to that obtained by phylogenetic analysis. We hypothesize that the Rop groups represent classes with distinct functions, which are specified by the unique protein sequence elements in each group and by their distinct expression patterns.

Detoxification of mycotoxins in planta as a strategy for improving grain quality and disease resistance: identification of fumonisin-degrading microbes from maize

Fumonisins are mycotoxins produced by Fusarium moniliforme and several related species associated with ear molds in maize. They consist of a long chain amine alcohol with two ester-linked tricarballylate groups, and their biological activity is likely through disruption of sphingolipid biosynthesis. Fumonisins are of concern due to their toxicity to livestock (especially horses) and possible carcinogenicity, and are currently under review for possible regulation by the United States Food and Drug Administration. Fusarium moniliforme is almost ubiquitous in field-grown maize, so the impact of this toxin on the food chain is potentially quite large. Fumonisins are also phytotoxic and some evidence links them to virulence of maize isolates of F. moniliforme. As a first step towards engineering maize to detoxify fumonisins, we have isolated microbes from field-grown, moldy maize kernels and stalk tissue, that are capable of growing on fumonisin B1 as a sole carbon source. Two species, identified as Exophiala spinifera and Rhinocladiella atrovirens, belong to the “black yeasts” found widely in plant debris. Interestingly, several E. spinifera isolates, collected outside the US. from non-maize sources, were also found to metabolize fumonisins. A gram (-) bacterium from stalk tissue, isolate 2412.1, also metabolized fumonisin in liquid culture. Both fungal and bacterial isolates produce 14CO2 from uniformly labeled fumonisin, indicating extensive metabolism of this important toxin. A soluble esterase activity capable of hydrolyzing fumonisin tricarballylate esters can be recovered from culture supernatants or lysates of these microbes. A soluble, heat-labile activity resulting in loss of a free primary amine group from AP1 has also been detected in cell lysates of E. spinifera 2141.10. Work is in progress to clone genes corresponding to these fumonisin degradative enzymes and express them in transgenic maize.

Transgenic crops for enhanced disease resistance and food safety.

Maize grain is subject to infection by a variety of pre-harvest molds, including Fusarium verticillioides (formerly F. moniliforme), a major causal agent of Fusarium ear mold of maize. F. verticillioides produces fumonisins, a family of mycotoxins of concern due to their acute toxicity to certain livestock, and their potential carcinogenicity (Dmello et al., 1999). Fumonisins inhibit sphingolipid biosynthesis in a wide range of organisms, but their mode of action as toxins and carcinogens is not fully understood. Incorporating natural ear mold resistance into elite maize germplasm has been difficult, due to complexity of the trait and the strong environmental component to ear mold symptom development, which complicates screening germplasm.