Defensive genes in plants can be activated by several different types of nonpeptide signaling molecules. An endogenous polypeptide, consisting of 18 amino acids, was isolated from tomato leaves and was able at very low concentrations to induce the synthesis of two wound-inducible proteinase inhibitor proteins when supplied to young tomato plants. The sequence of the polypeptide was determined, and an identical polypeptide was synthesized that possessed full inducing activity. These data establish that a polypeptide factor can initiate signal transduction to regulate the synthesis of defensive proteins in plant tissues.

endogenous elicitor ͉ plant defense ͉ defensin ͉ hydrogen peroxide S imilarities have been noted among early signaling components of animal and plant innate immune systems, including leucine-rich repeat receptor-mediated recognition of pathogenassociated molecular patterns and͞or elicitors from pathogens and the resulting activation of defense gene transcription involved in early steps of immunity (1-14). Several peptides originating from pathogens can activate the plant innate immune response, including fungal elicitors Pep13, AVR9, and elicitins (1-3), and bacterial elicitors hrpZ, NPP1, flg22, and elf13 (4-7). We report here that a 23-aa peptide, isolated from extracts of Arabidopsis leaves and called AtPep1, exhibits characteristics of an endogenous elicitor of the innate immune response. Endogenous plant peptides that activate genes specifically for defense against pathogens have not been reported previously to our knowledge, although systemin peptides, which are found only in Solanaceae species, activate antiherbivore defense genes. AtPep1 was first identified in soluble extracts of Arabidopsis leaves by its ability, at subnanomolar concentrations, to cause an alkalinization of the medium of suspension cultured cells, a typical response of cell cultures to peptide elicitors (15-19). AtPep1 is derived from the C terminus of a 92-aa precursor protein AtproPep1. The peptide activates the transcription of defensin, a gene extensively studied for its role in innate immunity in Arabidopsis, the production of H 2 O 2 , and the expression of PROPEP1. Constitutive overexpression of PROPEP1 confers resistance against a root pathogen Pythium irregulare. PROPEP1 orthologs are found in numerous important agricultural crop species, including both dicots and monocots, and may provide novel genes for investigating crop productivity. Results and DiscussionAtPep1 was purified to homogeneity ( Fig. 1 A and B) and characterized as a peptide by its molecular mass (Fig. 1C) and amino acid sequence (Fig. 1D), which together indicated that the peptide was not posttranslationally modified. Chemically synthesized AtPep1 was found to be as active as native AtPep1, having a half-maximal activity of Ϸ0.25 nM in the alkalinization assay. Peptides present in an 1% trifluoroacetic acid͞water extract of Arabidopsis tissues were passed through a reverse-phase semipreparative C18 flash chromatography column and separated on a G-25 Sepharose column as described in Materials and Methods. The breakthrough peak was applied to a C18 HPLC column, and 10 l from 2-ml fractions from the column was assayed for alkalinization activity. (B) The peak identified in A as AtPep1 was further purified through two additional chromatography steps and finally purified by narrow-bore HPLC as described in Materials and Methods. Fractions were assayed as in A. The active peak is identified with arrows. (C) Analysis of the biologically active peak by MALDI-MS. (D) The amino acid sequence of the purified peptide, determined by Edman degradation. The daltons calculat...

A 5-kDa polypeptide was isolated from tobacco leaves that induced a rapid alkalinization of the culture medium of tobacco suspension-cultured cells and a concomitant activation of an intracellular mitogen-activated protein kinase. An N-terminal sequence was obtained, and a cDNA coding for the 49-aa polypeptide was isolated from a tobacco cDNA library. The cDNA encoded a preproprotein of 115 amino acids that contained the polypeptide at its C terminus. A search among known expressed sequence tags revealed that genes encoding Rapid ALkalinization Factor (RALF) preproproteins were present in various tissues and organs from 16 species of plants representing 9 families. A tomato homolog of the polypeptide was synthesized and, when supplied to germinating tomato and Arabidopsis seeds, it caused an arrest of root growth and development. Although its specific role in growth has not been established, the polypeptide joins the ranks of the increasing number of polypeptide hormones that are known to regulate plant stress, growth, and development

leucine-rich repeat receptor kinase ͉ pathogen defense ͉ photoaffinity labeling ͉ microsomal membranes

A gene that encodes systemin, a mobile 18-amino acid polypeptide inducer of proteinase inhibitor synthesis in tomato and potato leaves, has been isolated from tomato, Lycopersicon esculentum. Induction of proteinase inhibitors in plants is a response to insect or pathogen attacks. The gene has 10 introns and 11 exons, ten of which are organized as five homologous pairs with an unrelated sequence in the eleventh, encoding systemin. Systemin is proteolytically processed from a 200-amino acid precursor protein, prosystemin. Prosystemin messenger RNA was found in all organs of the plant except the roots and was systemically wound-inducible in leaves. Tomato plants transformed with an antisense prosystemin complementary DNA exhibited greatly suppressed systemic wound induction of proteinase Inhibitor I and II synthesis in leaves.

Some animal and yeast hormone genes produce prohormone polypeptides that are proteolytically processed to produce multiple copies of hormones with the same or different functions. In plants, four polypeptides have been identified that can be classed as hormones (intercellular chemical messengers) but none are known to be produced as multiple copies from a single precursor. Here we describe a polyprotein hormone precursor, present in tobacco plants, that gives rise to two polypeptide hormones, as often found in animals and yeast. The tobacco polypeptides activate the synthesis of defensive proteinase-inhibitor proteins in a manner similar to that of systemin, an 18-amino-acid polypeptide found in tomato plants. The two tobacco polypeptides are derived from each end of a 165-amino-acid precursor that bears no homology to tomato prosystemin. The data show that structurally diverse polypeptide hormones in different plant species can serve similar signalling roles, a condition not found in animals or yeast.

Insect-induced defenses occur in nearly all plants and are regulated by conserved signaling pathways. As the first described plant peptide signal, systemin regulates antiherbivore defenses in the Solanaceae, but in other plant families, peptides with analogous activity have remained elusive. In the current study, we demonstrate that a member of the maize ( Zea mays ) plant elicitor peptide (Pep) family, ZmPep3, regulates responses against herbivores. Consistent with being a signal, expression of the ZmPROPEP3 precursor gene is rapidly induced by Spodoptera exigua oral secretions. At concentrations starting at 5 pmol per leaf, ZmPep3 stimulates production of jasmonic acid, ethylene, and increased expression of genes encoding proteins associated with herbivory defense. These include proteinase inhibitors and biosynthetic enzymes for production of volatile terpenes and benzoxazinoids. In accordance with gene expression data, plants treated with ZmPep3 emit volatiles similar to those from plants subjected to herbivory. ZmPep3-treated plants also exhibit induced accumulation of the benzoxazinoid phytoalexin 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside. Direct and indirect defenses induced by ZmPep3 contribute to resistance against S. exigua through significant reduction of larval growth and attraction of Cotesia marginiventris parasitoids. ZmPep3 activity is specific to Poaceous species; however, peptides derived from PROPEP orthologs identified in Solanaceous and Fabaceous plants also induce herbivory-associated volatiles in their respective species. These studies demonstrate that Peps are conserved signals across diverse plant families regulating antiherbivore defenses and are likely to be the missing functional homologs of systemin outside of the Solanaceae.

Tomato Tomato Transformation. The prosystemin expression construct was introduced into Agrobacterium tumefaciens strain LA 4404 and was used to transform tomato (var. Better Boy) cotyledon tissue as previously described (10).Grafting Experiments. Plants (Better Boy) were used 5-6 weeks after germination. The upper halves of the plants were excised at the midpoint of the stem and all the leaves were trimmed away with the exception of the pair of leaves immediately beneath the apical meristem. The cut ends ofthe stems were notched and grafting was accomplished by aligning the notched ends of the stems and wrapping the graft site with Parafilm. Grafted plants were enclosed in a transparent plastic bag in the laboratory for 4 or 5 days before being allowed to regenerate and grow in the greenhouse for 7-8 weeks. Senescent leaves on the lower half of each grafted plant were periodically removed, although the plants were left undisturbed for at least 1 week prior to assaying the levels of proteinase inhibitors. Wounding and Immunoiogical Assay of Proteinase Inhibitors. Wounding experiments utilized 14-to 16-day-old plants, which were wounded on the lower of the two primary leaves and left under constant illumination for 24 hr. The levels of proteinase inhibitor I and inhibitor II were measured as described (11,12).