Laboratory of Ann Hirsch

Molecular and Genetic Analysis of Plant-Microbe Interactions.

The research in my lab focuses on the early stages of the interaction between nitrogen-fixing Rhizobium bacteria and legumes such as alfalfa, pea, and soybean in order to determine why this interaction occurs exclusively with leguminous plants. Root nodules are formed following the perception of signal molecules (lipochitooligosaccharides) from a compatible Rhizobium. The nodules house the bacteria, which convert atmospheric nitrogen into ammonia (Fig.1).

Our investigations focus mainly on the earliest stages in the symbiosis, especially attachment. We have been studying the plant's role in establishing the symbiosis by investigating the involvement of both lectin and early nodulin (ENOD) genes. We have approached the study of lectin function in nodulation by doing both gain-of-function and loss-of-function (antisense technology and RNAi) experiments.

The plant's role can also be studied by looking at single-gene plant mutants. We are studying Lotus japonicus and Melilotus alba, two autogamous diploid legumes. In M. alba, A number of mutants, which are defective in both the nitrogen-fixing and mycorrhizal symbioses, have already been identified and characterized. One of these mutants, sym3, forms neither root nodules nor mycorrhizae (Fig. 2). We are collaboration with Dr. Akihiro Suzuki of Saga University in Japan on L. japonicus.

On the Rhizobium side, we are studying rhizobial attachment by using an in vitro assay for biofilm formation. Biofilms are structured communities of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface; they have clinical, industrial and environmental impacts. The in vitro assay allows us to screen a large number of mutants and thus serves as an excellent model system for studying root colonization (Fig. 3).

A new project is on studying the beta-rhizobial strain Burkholderia tuberum, which also nodulates and fixes nitrogen with legume hosts. Current research involves the sequecing of four plant-associated Burkholderia genomes as well as a search for B. tuberum mutants (Fig. 4).

We also study other plant-microbe interactions. One of these is the Frankia-actinorhizal plant symbiosis. Frankia is a Gram-positive actinomycete that fixes nitrogen both within the nodule and ex planta (Fig. 5). We have a long-standing collaboration with Dr. Maria Valdés of IPN in Mexico City investigating a novel group of diazotrophs isolated from nodules of Casuarina growing in Mexico. These bacteria have nifH, but they are not Frankia. Phylogenetic studies indicate that some of the isolates belong to the Micromonosporaceae. Current research involves the sequencing of the nitrogen-fixing strain and also M. aurantiaca.