Acknowledgement

This work is supported in part by grants from National Natural Science Foundation of China (grants 39925017 and 39970168), the Ministry of Education of China and the 863 program from the Ministry of Science and Technology of China.

GENETIC AND FUNCTIONAL ANALYSIS OF NOVEL RHIZOBIUM TROPICICIAT899 SYMBIOSIS-RELEVANT LOCI IDENTIFIED THROUGH Tn5 INSERTION MUTAGENESIS AND SELECTION OF ACID-SENSITIVE MUTANTS ON ACIDIFIED SOLID MEDIA

P. Vinuesa1'3, F. Neumann-Silkow1, A. Aranda-Rickert1, C. Pacios-Bras2, H. Steele1,

!FB Biologie, Dept. of Cell Bio and Applied Botany, Philipps-University, Germany

2Leiden University, Institute for Mol. Plant Sei., The Netherlands

3Centro de Investigación sobre Fijación de Nitrógeno, UNAM, Mexico

Research on the genetic and physiological basis of acid tolerance in rhizobia is both of practical and fundamental significance. Firstly, acid soils constrain nodulation and N2-fixation in many legume-Rhizobium symbioses of agronomic interest (Glenn et al. 1999). Since both the micro- and the macrosymbiont can represent the acid-sensitive component of the symbiosis, investigations aimed at finding acid-tolerant plant germplasm and compatible rhizobial strains are of utmost agronomic and ecological relevance (Zahran et al. 1999). Secondly, rhizobia, as intracellular microsymbionts, have been postulated to inhabit an acidic lytic compartment, the symbiosome, which resembles in several aspects the phagosomes inhabited by mammalian intracellular pathogens (Mellor et al. 1989; Parniske 2000). As a corollary, it can be hypothesized that the capacity of the microsymbiont to adapt to an acidic environment may be of fundamental importance for host cell invasion and the consequent establishment of a N2-fixing Rhizobiwn-legumc symbiosis. The aim of this study was to identify novel rhizobial genes required for acid tolerance, and eventually also for symbiosis. For this purpose R. tropici CIAT899 was chosen as the model organism, since this strain is well known to be highly tolerant to several environmental stress factors, including acidity, being able to grow on media acidified down to pH 4.0 (Graham et al. 1994). Four prototrophic acid-sensitive Tn5 insertion mutants of Rhizobium tropici CIAT899 were identified by plating a mutant bank on acidified minimal medium (pH 4.2). These mutants (899-PV1, 899-PV2, 899-PV4, 899-PV9) elicit Ndv" and Fix" nodules on bean plants. Light and electron microscopy of the nodules elicited by strains 899-PV4 and 899-PV9 showed that they can invade nodules. However, bacteroids are not efficiently released from infection threads, and do not elongate. Very few symbiosomes are found within highly vacuolated host cells, lysing prematurely, indicating that the mutated loci affect bacteroid intracellular accommodation and nodule differentiation. Cosmids complementing the defects displayed by the 4 strains could be isolated. The sequence of cosmid subclones complementing 899-PV4 and 899-PV9 was determined, resulting in the identification of two novel symbiosis-relevant loci. The Tn5 insertions in strains 899-PV4 and 899-PV9 were mapped by sequencing. The former disrupted a gene with high sequence identity to the Agrobacterium tumefaciens chromosomal virulence locus acvB (Vinuesa et al. submitted). We demonstrated by means of reciprocal complementation studies, that the agrobacterial and rhizobial acvB genes are orthologs. Biological basis of the acid tolerance displayed by these mutants is presently not understood.

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