Journal of Experimental Botany, Vol. 52, No. 362, pp. 1947-1948,
September 1, 2001
© 2001 Oxford University Press
Gene Note |
Cryptogein affects expression of 
3, 6 and ß1 20S proteasome subunits encoding genes in tobacco
UMR INRA-Université de Bourgogne, Phytopharmacie et Biochimie des Interactions Cellulaires, INRA BP 86510, 21065 Dijon cedex, France
Received 26 April 2001; Accepted 4 June 2001
Abstract
Twelve 
and ß 20S proteasome subunits cDNAs showing 70–82% identity with the corresponding genes in Arabidopsis or rice, and features of eukaryotic proteasome subunits were cloned in tobacco. Only ß1-tcI 7, 3 and 6, 20S proteasome subunits encoding genes were up-regulated by cryptogein, a proteinaceous elicitor of plant defence reactions. These results led to the hypothesis that the activation of ß1-tcI 7, 3 and 6 could induce a specific proteolysis involved in the hypersensitive response and systemic acquired resistance monitored by cryptogein.
Key words: Elicitins, 20S proteasome subunits, plant defence reactions.
In eukaryotes, the 26S proteasome is the central multicatalytic proteinase complex comprising two subcomplexes: the 20S core particle that performs proteolysis and the 19S regulatory particle that recognizes the protein targeted for degradation. The 20S proteasome is a stack of four seven-membered rings, the two outer rings being formed by seven subunits and the two inner rings by seven ß subunits. The central ß rings enclose a cavity that houses the active sites allowing the progressive degradation of proteins. In mammals, proteasomes are shown to be involved in the degradation of misfolded proteins and also in biological functions such as cell cycle progression or cell death, and specific proteolysis such as the cleavage of propeptides in order to activate peptides or allowing the antigene presentation (for a review see Coux et al., 1996
).
In plants, the 20S proteasome was shown to be involved in cell cycle progression (Genschik et al., 1994), in senescence (Bahrami and Gray, 1999), in the early stages of seedling development (Ito et al., 1999), and in defence responses (Becker et al., 2000). Since it was previously shown that tcI 7, a gene encoding a ß1 proteasome subunit, was up-regulated by cryptogein, a proteinaceous elicitor of the defence reaction (Petitot et al., 1997; Etienne et al., 2000), it was interesting to investigate how genes encoding other proteasome subunits are regulated during the initiation of defence reactions. The published sequences of Arabidopsis thaliana (Parmentier et al., 1997) and rice (Oryza sativa) (Sassa et al., 2000) and ß subunits were used to determine degenerated primers for RT-PCR and RACE. Reverse transcription was performed using the ‘First strand cDNA synthesis kit for RT-PCR’ (Boehringer) with 3 µg total RNA. RACE amplifications were performed from 1 µg poly(A)+ using the ‘MarathonTM cDNA Amplification kit’ (Clontech). The cDNAs were cloned in pGEM-Teasy vector (Promega, France) and sequenced (Genome Express, France). Sequences of all obtained cDNAs were submitted to the EMBL database. The corresponding genes were named: NtPA1 (100% identity with NtPSA1, Y16644, previously isolated from tobacco; Bahrami and Gray, 1999), NtPA3 (AJ291733), NtPA4 (AJ291734), NtPA5 (AJ291735), NtPA6 (AJ291737), and NtPA7 (AJ291738) for subunits and NtPB1 (=tcI 7, Y09505, Petitot et al., 1997), NtPB2 (AJ291736), NtPB3 (AJ291739), NtPB4 (AJ291740), NtPB5 (AJ291741), NtPB6 (AJ291742), and NtPB7 (AJ291743) for ß subunits. The amino acid sequence homologies between the tobacco and ß subunits and the corresponding genes in Arabidopsis thaliana and in rice are high (75–98%), confirming the characterization of cloned cDNAs as 20S proteasome or ß subunits.
Figure 1 shows the alignments of the deduced amino acid sequences of the (Fig. 1A) or ß (Fig. 1B) subunit family of tobacco. Four of the six characterized subunits (NtPA1, NtPA3, NtPA6, NtPA7) were found to have the -type signature at their N-termini and two (NtPA6 and NtPA7) showed a Tyr residue (Y), essential for the assembly of the ring (Groll et al., 1997). Moreover, the amino acid sequences of NtPA1, NtPA3 and NtPA4 show a putative nuclear localization signal (NLS), with a typical KKXXXK sequence (Tanaka et al., 1990). The ß-type signature was conserved in all of the cloned tobacco ß subunits. In animals, ß1, ß2 and ß5 were previously shown to be processed to remove their propeptides (Chen and Hochstrasser, 1996), and their N terminal Thr residues to form the catalytic site of the 20S proteasome (Groll et al., 1997). The sequence of tobacco ß1-tcI 7 (NtPB1) and ß2 (NtPB2) show such a propeptide and a Thr residue (T) preceded by a Gly (G), the Gly/ Thr pair representing the cleavage site (Groll et al., 1997). The sequence analysis of tobacco 20S proteasome subunits also shows the presence of conserved glycine residues (Chen and Hochstrasser, 1996). Thus, the sequences obtained in this study showed sufficient characteristics of or ß proteasome 20S subunits to allow the analysis of the expression of the various and ß proteasome subunits during the induction of plant defence mechanisms monitored by cryptogein in tobacco.
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Figure 2
shows the kinetics of accumulation of mRNA corresponding to and ß 20S proteasome subunits: for 1, 4, 5, 7, ß2, ß3, ß4, ß5, ß6, and ß7 proteasome subunits, the transcripts remained undetected. A high accumulation of transcripts was observed from 9 h or 12 h only for 3, 6 and ß1-tcI 7 and hypersensitive-like lesions appeared within 16–24 h after the treatment. A treatment of leaves with bovine serum albumin instead of cryptogein confirmed that infiltration of a foreign protein was not sufficient to induce any proteasome subunits (not shown). These data indicate a specific induction of ß1, 3 and 6 subunits of the 20S proteasome encoding genes, which begins before the appearance of the hypersensitive response and correlated with the induction of systemic acquired resistance monitored by cryptogein. It could be possible that during the induction of defence reactions, the incorporation of elicitin-stimulated subunits into the proteasome structure leads to the replacement of constitutive corresponding subunits, as previously observed in ‘immunoproteasomes’ in animals. In mammalian cells, three ß subunits X, Y and Z were replaced by other interferon-induced ß subunits named LMP7, LMP2 and MECL1 modifying the proteolytic activities of these ‘immunoproteasomes’ (Eleuteri et al., 1997). The results obtained in this work indicate specific activation of 3, 6 and ß1 subunits, probably in order to monitor specific proteolysis involved in defence reactions. Only further detailed multiparametric studies will lead to an understanding of their different putative functions.
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Acknowledgments
This work is supported by INRA and by the ‘Conseil Régional de Bourgogne’. Ph Etienne is supported by a grant from the ‘Ministère de l'Education Nationale, de la Recherche et de la Technologie’ and Anne-Sophie Petitot by Genoplante.
Notes
1 To whom correspondence should be addressed. Fax: +33 3 80 69 32 65. E-mail: suty{at}epoisses.inra.fr 
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