The involvement of a cysteine proteinase in the nodule development in Chinese milk vetch infected with Mesorhizobium huakuii subsp. rengei

Abstract

Cys proteinases play important roles in plant cell development and senescence. A cDNA, AsNODf32, obtained by differential screening of a nodule cDNA library of the leguminous plant Chinese milk vetch (Astragalus sinicus), represents a nodule-specific Cys proteinase similar to that reported for the actinorhizal Alnus glutinosa-Flankia symbiosis. A characteristic feature of this proteinase is the presence of a putative vacuolar targetting signal, LQDA, within its propeptide. Expression of the AsNODf32 gene, which was studied on northern blots and in situ hybridization, showed good correlation with the onset of nodule senescence. In situ hybridization studies revealed that AsNODf32 was expressed in senescent-infected tissue at the base of the nodule, as well as in interzone II-III of the infected nodules. In addition to degrading old nodule tissues and bacteroids, AsNODf32 protein may be required as a component of tissue remodeling during nodule development.

Figure 1

Detection of a nodule-specific DNA fragment by differential display. cDNAs synthesized along mRNAs isolated from either nodules, leaves, or roots of Chinese milk vetch were used as templates in PCR with a combination of A21 and A22 oligonucleotide primers (Bex). The PCR products were separated by agarose gel electrophoresis and compared with each other. Arrow indicates a 148-bp nodule-specific band (f32). Molecular markers are λ-DNA- digested with StyI.

Figure 2

Alignment of the deduced amino acid sequence of AsNODf32 with those of Cys proteinases of the papain super family. Dots represent identical amino acid residues. Dashes represent blanks. Amino acid residues (AsNODf32 numbering) involved in catalysis (C-149 and H-286), active-site formation (Q-143, N-307, S-308, and W-309), and the disulfide bridges (C-146/C-189, C-180/C-222, and C-280/C-322) are underlined. A putative vacuole-targeting signal (LQDA) and the ER-targeting signal (KDEL) are also indicated by dotted underlines, respectively. As, Chinese milk vetch; Ag, A. glutinosa (Goetting-Minesky and Mullin, 1994); Pm, P. menziesii (Tranbarger and Misra, 1996); Ps, Pisum sativum (pea; Kardailsky and Brewin, 1996); Pv, P. vulgaris (Sohlberg and Sussex, 1997); Vm, Vigna mungo (Akasofu et al., 1989); Vs, vetch (Becker et al., 1997).

Figure 3

Southern-blot analysis of Chinese milk vetch (A.s) and M. huakuii (M.h) genomic DNAs. Restriction enzymes used to digest DNA are as indicated. Probes used were a 148-bp N-terminal fragment (A), a 453-bp C-terminal fragment (B), and an entire region (1,291 bp, C) of AsNODf322 cDNA. Sequences used to generate probes A, B, and C are illustrated in the bottom section, relative to a restriction map of AsNODf32 cDNA. Arrows indicate the position of lambda DNA fragments digested with HindIII.

Figure 4

Northern-blot analysis to detect AsNODf32 transcripts. Total RNA isolated from various tissues of Chinese milk vetch was hybridized with AsNODf32 cDNA as a probe. Arrows indicate positions of RNA size markers (Boehringer Mannheim, Basel). For control, the same filter was reprobed with the gene for β-tubulin (bottom).

Figure 5

In situ hybridization analysis of the AsNODf32 gene expression. Median longitudinal sections of Chinese milk vetch nodules infected with M. huakuii (30 d post-infection) were hybridized with riboprobes containing digoxygenin. Hybridization signal was detected as a blue precipitate by staining with BCIP and NBT. A, Schematic representation of the five distinct regions (Vasse et al., 1990; Mylona et al., 1995) of an indeterminate type nodule of Chinese milk vetch. B, Nodule section hybridized with AsNODf32 antisense strand RNA probe. C, Nodule section hybridized with AsNODf32 sense strand RNA as the probe.