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For a Nodule-Specific Cysteine Proteinase (Nitrogen Rixation/Ahlus Gluinosa/Differential Screening/Nodulin) M

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For a Nodule-Specific Cysteine Proteinase (Nitrogen Rixation/Ahlus Gluinosa/Differential Screening/Nodulin) M Proc. Nati. Acad. Sci. USA Vol. 91, pp. 9891-9895, October 1994 Plant Biology Differential gene expression in an actinorhizal symbiosis: Evidence for a nodule-specific cysteine proteinase (nitrogen rixation/Ahlus gluinosa/differential screening/nodulin) M. P. GOETTING-MINESKY AND B. C. MULLIN Graduate Program in Plant Physiology and Genetics, Department of Botany, Center for Legume Research, University of Tennessee, Knoxville, TN 37996-1100 Communicated by Robert H. Burris, July 5, 1994 ABSTRACT Nodules formed on the roots of actinorhizal nitrogen. Alteration of lateral root morphology by the sym- plants as a consequence of nitrogen-fixing symbioses with the biosis results in the characteristic lobed nodules ofAlnus (3, actinomycete Frankia appear to result from modification ofthe 4). developmental pathway that leads to lateral root formation. In the Rhizobium4egume symbioses, plant proteins ex- Presently no information exists about factors that control this pressed specifically in nitrogen-fixing root nodules have been developmental switch or, until now, about genes that are observed and classified, according to time of expression, as differentially expressed as a result of an altered developmental early and late nodulins (5). Early nodulins, thought to func- pathway. Differential screening of an Alnus glutinosa nodule tion in bacterial invasion of the root and in nodule organo- cDNA library revealed altered levels of gene expression in genesis, may be expressed as a host response to one or more nodules as compared with roots and allowed isolation of host bacterial signals or as a response to altered host hormone plant nodule-specific cDNA sequences. The deduced amino levels. Late nodulins are considered to be involved in estab- acid sequence of one full-length cDNA, AgNOD-CP1, repre- lishment and maintenance ofa nodule environment favorable sents a nodule-specific cysteine proteinase similar to cysteine for nitrogen reduction and ammonium assimilation and may proteinases of the papain superfamily. Residues critical to be expressed in response to changing levels of nodule me- catalysis, active site, and disulfide bridges are conserved. tabolites or oxygen (6). Suggested roles for this enzyme are as a defense response to Little is known about the biochemistry and molecular Frankia invasion, as a component of tissue remodeling in root biology of the infection process and nodule formation in and nodule tissues, as a cell cycle component, or as an element actinorhizal systems. Indeed, it has been suggested that, of protein turnover. Complexity of hybridization patterns because actinorhizal nodules are derivatives of lateral root that the for primordia rather than new organs as in the Rhizobium- revealed by Southern blot analysis suggests gene legume symbioses, proteins analogous to legume nodulins AgNOD-CP1 is a member of a multigene family. Northern may not exist in Frankia-induced nodules. Host plant gene hybridization results indicate that this gene may have been expression in actinorhizal nodules has not been studied and recruited for a role specific to this symbiosis, a phenomenon the occurrence of nodulins has not been demonstrated (7, 8). observed in the Rhizobium-legume symbioses, perhaps com- We report evidence of nodule-specific gene expression in mon to many microbe-plant interactions. an actinorhizal plant, Alnus glutinosa, the European black alder. Extensive variation in levels of gene expression be- Actinorhizal plants form a group of primarily woody an- tween root and nodule tissues was revealed and a number of giosperms capable of participating in a mutually beneficial nodule-specific cDNA clones were isolated. The deduced symbiosis with the nitrogen-fixing actinomycete Frankia. amino acid sequences of two of these cDNA sequences have Although traditionally viewed as being phylogenetically di- been determined.* One is a nodule-specific glycine- and verse, recent phylogenies based on molecular data indicate histidine-rich protein (9). The other, the subject ofthis paper, that these plants are much more closely related than previ- represents the sequence of a nodule-specific cysteine pro- ously thought (1). Ecologically, actinorhizal plants are ver- teinase which is similar in sequence to cysteine proteinases satile pioneers that can be components of primary succes- of both plants and animals. sions on exposed sites or of secondary successions on disturbed sites, where they facilitate the entry of new species by increasing the soil nitrogen content (2). MATERIALS AND METHODS In actinorhizal systems, presence of a compatible Frankia Bacterial Culture and Plant Material. Frankia strain strain alters the developmental path of lateral root formation WgAvcI1 was maintained in pure culture in Frankia broth so that those roots become nitrogen-fixing nodules and serve (10). One- to 2-week-old cultures were harvested and used to as a source of reduced nitrogen for the host plant. The inoculate A. glutinosa seedlings (11). infection process is believed to be initiated in response to A. glutinosa seedlings were grown as described (12). some diffusible signal produced by Frankia. A few hours Nodule and root tissues were collected for RNA extractions after contact with Frankia, deformation ofgrowing root hairs at various stages of development from Frankia-inoculated is the first visible indication ofpossible nodule formation. As and uninoculated seedlings, respectively, during times of bacterial filaments penetrate the root hair, cortical cell divi- peak nitrogenase activity (13). Young leaves were collected sions lead to formation of a prenodule which is colonized by for DNA extraction from A. glutinosa after a 1- to 1.5-day Frankia. Lateral root growth is induced in the pericycle; the dark treatment of the trees. All tissues were collected onto primordium ofthis root penetrates the infected prenodule and ice, weighed, frozen in liquid nitrogen, and stored at -80°C becomes infected by Frankia. The microsymbiont rapidly until needed. proliferates within host cells and begins to fix atmospheric Nucleic Acid Extractions. A modification (12) of a plant DNA minipreparation protocol (14) was used to recover total The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" *The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. U13940). 9891 Downloaded by guest on October 3, 2021 9892 Plant Biology: Goetting-Minesky and Mullin Proc. Natl. Acad Sci. USA 91 (1994) RNA from A. glutinosa roots and nodules. Placental RNase ical), M13 reverse primer (United States Biochemical) and an inhibitor (RNasin; Promega) was added (625 units/ml) to internal 26-nt oligomer were used as sequencing primers. each RNA sample. The integrity of each RNA sample was Autoradiographs of sequencing gels were read manually and evaluated after electrophoresis through agarose gel (15) data were entered into the SEQED program for further DNA and/or formaldehyde/agarose gel (16). Poly(A)+ RNA for sequence analysis by the University of Wisconsin Genetics 32P-labeled cDNA probes was selected from total RNA that Computer Group sequence analysis software on the VAX had been selectively precipitated with 2 M LiCl from a total cluster at the University of Tennessee, Knoxville (23, 24). nucleic acid preparation by a modification (12) ofthe method The deduced amino acid sequence ofthe nucleotide sequence in ref. 17. (TRANSLATE) was compared with GenBank and EMBL se- Total genomic DNA was extracted from the leaves of A. quence data banks by the TFASTA program. glutinosa according to the same plant DNA minipreparation (12, 14). Before digestion with selected restriction endonu- cleases, contaminating RNA was hydrolyzed with RNase A RESULTS and RNase T1. Plasmid DNAs were recovered from bacterial Nodule cDNA Library Construction and Screening. The titer cultures by an alkaline lysis minipreparation protocol (15). of the A. glutinosa nodule cDNA library was 1.19 x 1010 Total genomic DNA was extracted from pure cultures of colony-forming units per milliliter. Contamination of this Frankia strain WgAvcI1 by a small-scale CTAB (hexadecyl- cDNA library by DNA copies of rRNA was <0.5%. The trimethylammonium bromide) extraction procedure (18). average size ofthe nodule cDNA inserts in pcDNAII was =1 This DNA was also treated with RNase A and RNase T1. kb. Differential screening of the nodule cDNA library indi- cDNA Synthesis, Library Construction, and cDNA Probe cated altered gene expression in the Alnus nodules when Preparation. Poly(A)+ RNA used for cDNA synthesis was compared with root gene expression. Some mRNAs ap- selected twice by oligo(dT)-cellulose affinity column chro- peared to result from nodule-specific or -enhanced gene matography, initially from total RNA preparations from roots expression. The presence of other transcripts indicated or nodules (12, 17, 19). RNasin (625 units/ml) was added to equivalent expression levels of some genes in both root and poly(A)+ RNA-containing fractions identified by agarose gel nodule tissues whereas other genes showed lower expression electrophoresis. in nodules than in roots. A total of 582 putative nodule- The nodule cDNA library was prepared from A. glutinosa specific clones were stored as frozen cultures. The charac- nodule poly(A)+ RNA with Librarian II (version 2.4; Invit- terization of one of these putative nodule-specific clones, rogen)
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