A subtilisin-like protein from soybean contains an embedded, cryptic signal that activates defense- related genes Gregory Pearcea,1,2, Yube Yamaguchia,b,1,3, Guido Baronaa, and Clarence A. Ryana,4 aInstitute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340; and bLaboratory of Crop Physiology, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan Edited* by Rodney B. Croteau, Washington State University, Pullman, WA, and approved July 9, 2010 (received for review June 1, 2010) Among the arsenal of plant-derived compounds activated upon onate signaling pathway, producing protease inhibitors and other attack by herbivores and pathogens are small peptides that initiate defense compounds that protect the plant from further attack (14). and amplify defense responses. However, only a handful of plant Relatively few endogenous peptide defense signals have been signaling peptides have been reported. Here, we have isolated a 12- isolated thus far. These include a family of glycopeptides from the aa peptide from soybean (Glycine max) leaves that causes a pH in- Solanaceae that are functionally related to systemin, hydrox- crease of soybean suspension-cultured cell media within 10 min at yproline-rich glycopeptide systemins (HypSys) (15), and a family low nanomolar concentrations, a response that is typical of other of signaling peptides from Arabidopsis (AtPeps) that amplify the endogenous peptide elicitors and pathogen-derived elicitors. The innate immune responses through the jasmonate/ethylene and amino acid sequence was determined and was found to be derived salicylate signaling pathways (16, 17). Another plant peptide sig- from a member of the subtilisin-like protease (subtilase) family. The nal generated from a plant has been found that is derived from an sequence of the peptide was located within a region of the protein intracellular protein. When cowpea (Vigna unguiculata) leaves that is unique to subtilases in legume plants and not found within were consumed by armyworm larvae, a proteolyzed fragment of any other plant subtilases thus far identified. We have named this the cowpea chloroplastic ATP synthase was produced in the insect peptide signal Glycine max Subtilase Peptide (GmSubPep). The gene gut that was found to elicit defense responses when deposited on (Glyma18g48580) was expressed in all actively growing tissues of the leaves in the oral secretions (18). The peptide was termed the soybean plant. Although transcription of Glyma18g48580 was inceptin and is an example of an indirect signal generated by the not induced by wounding, methyl jasmonate, methyl salicylate, or insect that initiates a specific plant defense response. ethephon, synthetic GmSubPep peptide, when supplied to soybean The complexity of the host–herbivore/pathogen relationship is cultures, induced the expression of known defense-related genes, becoming a common theme in plant biology. In recent work with such as Cyp93A1, Chib-1b, PDR12,andachs. GmSubPep is a unique inceptin, the peptide was active in inducing defense responses in plant defense peptide signal, cryptically embedded within a plant cowpea and not in another member of the Fabaceae family, protein with an independent metabolic role, providing insights into Glycine max (19). This is similar to systemin, which was found only plant defense mechanisms. in one clade of the Solanaceae. In a continuing search for plant elicitors of defense responses, we have isolated a 12-aa peptide damaged-self recognition | plant defense | signaling peptide | subtilase from Glycine max that induces the expression of defense genes. The peptide is processed from a unique region of an extracellular he modification of survival mechanisms during coevolution of subtilisin-like protease (subtilase), providing insight into the Tplant hosts with their biotic attackers resulted in the present- mechanism by which host plant–derived, damage-associated sig- day complexity of plant–pathogen and plant–insect interactions. nals mediate immune responses. Whereas the invading species has developed methods of adhesion, PLANT BIOLOGY penetration, and feeding, the plant has evolved mechanisms for Results and Discussion perception of attack and activation of defense responses, based on In our investigations of defense peptides, a bioassay has been surveillance of its own tissue. Damaged-self recognition occurs used that takes advantage of a dramatic increase in pH of the when signaling molecules are released from damaged cells and media of suspension cells when a bioactive peptide binds to its perceived by plant receptors to elicit a defense response (1). These receptor (20–22). A crude peptide fraction obtained from soy- elicitors can be of a heterogeneous nature, such as cutin monomers bean leaves displayed the ability to alkalinize soybean suspension or cell wall fragments of various sizes (2–4), which exist in all plant cell media when separated on a C18 reversed-phase column (Fig. species, or they can be a more specific, fine-tuned signal, such as 1A). Along with large, late-eluting peaks corresponding in re- endogenous peptide signals, which may be limited to a single tention time to the previously reported rapid alkalinization fac- phylogenetic family, coevolving with a specific predator (5–7). Collectively, these diverse compounds are termed endogenous elicitors or damage-associated molecular patterns (DAMPs). Author contributions: G.P. and Y.Y. designed research; G.P., Y.Y., and G.B. performed Plants have also evolved receptor-mediated recognition systems research; C.A.R. contributed new reagents/analytic tools; G.P. and Y.Y. analyzed data; for fungal and microbial biochemicals from the sites of infection, and G.P. and Y.Y. wrote the paper. termed microbe-associated molecular patterns (MAMPs). These The authors declare no conflict of interest. signals include bacterial peptide fragments, such as flg22 and elf18 *This Direct Submission article had a prearranged editor. (8, 9), fungal peptide elicitors, such as Pep13, AVR9, and elicitins Freely available online through the PNAS open access option. (10–12), chitin fragments from fungal cell walls (13), and the 1G.P. and Y.Y. contributed equally to this work. heptaglucoside elicitor from the oomycete Phytophtora mega- 2To whom correspondence should be addressed. E-mail: [email protected]. sperma (3). MAMPs and DAMPs are perceived on the plant cell 3Present address: Laboratory of Crop Physiology, Graduate School of Agriculture, surface by receptors that transduce a signal intracellularly, initi- Hokkaido University, Sapporo 060-8589, Japan. ating a defense pathway. For example, upon wounding of tomato 4Deceased October 7, 2007. plants, the plant peptide signal systemin is released from its pre- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cursor and, through receptor-mediated events, initiates the jasm- 1073/pnas.1007568107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1007568107 PNAS | August 17, 2010 | vol. 107 | no. 33 | 14921–14925 Downloaded by guest on September 28, 2021 1.0 A 6.0 % A Acetonitrile 0.8 5.6 5.2 40 0.6 pH 4.8 20 pH 0.4 D 4.4 0 0.2 0 20 40 60 TIME (min) 0 6.2 -0.2 B 0102030 0.03 5.8 TIME(Min) pH 5.4 1.2 0.02 5.0 B 50 60 TIME (min) Methanol % 1.0 30 0.8 0.01 20 pH 0.6 Absorbance (210nm) 10 D 0.4 0 0 40 50 60 70 TIME (min) 0.2 0 100 1416.83 1224.3 0.025 0.25 2.5 25 250 C MS [nM] Peptide 80 60 Fig. 2. Alkalinization assay kinetics of the synthetic peptide. (A) Cultured 40 soybean cells [strain: PI 553039 (Davis)] were used 4 d after subculture. Ali- quots of peptide or H O(10μL) were added to 1 mL of suspension cells on an Intensity (%) Intensity 1078.16 1585.92 2 20 orbital shaker at 160 rpm. The pH of the suspension cell media was recorded 1289.18 0 at various time points. (B) Aliquots of peptide (10 μL) were added to 1 mL of 1000 1601.8 2200 Mass (m/z) cells at various concentrations, and the pH of the suspension cell media was recorded after 15 min. Experiments were done in triplicate from three separate flasks of soybean suspension cells. Error bars indicate SD. D +NTPPRRAKSRPH- 1416.83 Da fi Fig. 1. HPLC purification of GmSubPep from soybean leaf extracts. (A)A The peptide sequence identi ed in the soybean genome data- crude leaf extract (see Materials and Methods) was applied to a reversed- base was derived from a gene designated Glyma18g48580, which fl phase C18 semipreparative HPLC column in 0.1% tri uoroacetic acid/H2O is predicted to code for a subtilisin-like protease (subtilase). and eluted with an acetonitrile gradient. Fractions (2 mL) were collected, Therefore, we have named the peptide Glycine max Subtilase and 10-μL aliquots were assayed for alkalinizing activity. The bioactive Peptide (GmSubPep). Because the predicted coding sequence of – fi fraction (32 34) designated by the bar was pooled for further puri cation. Glyma18g48580 in the database was incomplete, a start codon was (B) After several HPLC purification steps, the active fraction was eluted from a narrow-bore C18 column with a methanol gradient as described in SI predicted from the genomic DNA sequence by comparisons with Materials and Methods and assayed for alkalinizing activity. The active other subtilases, and the full-length coding region of Gly- fraction (50–51) was pooled for mass spectral analysis. (C) MALDI mass ma18g48480 was amplified by RT-PCR and sequenced (Fig. S2). spectral analysis of the pooled fractions from Fig.1B contained a major mass Glyma18g48580 contains all of the characteristic domains found peak at 1,416.8 Da. (D) The 1,416.83 mass peak was subjected to MS/MS (Fig. in plant subtilases, including a signal sequence for secretion to the S1), and the fragmentation spectra revealed a 12-aa peptide.