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- Materials and Methods
- Supporting Information
Plants are frequently attacked by voracious insect herbivores, and consequently have evolved a broad range of defense mechanisms to combat these pests. Some of the most effective plant defense mechanisms combine physical traits such as trichomes, thorns and cuticles (Levin, 1973) with chemical substances that deter feeding and ‘poison’ the insects. Often these defenses are constitutively produced and always available to protect the plant from herbivory. Plants also employ inducible defenses that are activated in response to herbivore attack. Induced defenses include indirect defenses such as production of green leaf volatiles and other volatile organic compounds that attract natural enemies of the attacking insects, and direct defenses that rely on the accumulation of secondary metabolites, defensive proteins, and other toxic compounds (Howe & Jander, 2008). Toxic secondary metabolites include glucosinolates, benzoxazinoids, alkaloids, and phenolics and other compounds. In addition, herbivore-defensive proteins are typically induced in response to insect feeding. A major class of defensive proteins includes the protease inhibitors (PIs) that rapidly accumulate in potato and tomato after insect feeding (Green & Ryan, 1972). In the insect gut, PIs interact with gut proteases and thereby inhibit digestion. Transgenic plants expressing PI genes have detrimental effects on herbivore growth; however, insects often counter this defense by producing more or different digestive proteases (Chen, 2008; Howe & Jander, 2008; Zhu-Salzman et al., 2008). Plant oxidative enzymes such as polyphenol oxidases (PPOs), peroxidases, lipoxygenases, and ascorbate oxidases are also involved in plant defenses against insect herbivores (Chen, 2008). They oxidize essential nutrients or form electrophilic products that react with the nucleophilic amino acid side chains and lower plant nutritive value (Felton et al., 1994; Constabel, 1999; Chen, 2008). For example, it has been shown that a maize anionic peroxidase suppresses caterpillar growth when it was ectopically expressed in callus (Dowd et al., 2010). Besides these proteins, some proteases, such as maize insect resistance 1-cysteine protease (Mir1-CP) and leucine aminopeptidase (LAP), have been shown to defend plants against insect herbivores (Chao et al., 1999; Chen, 2008; Zhu-Salzman et al., 2008).
Herbivore- or jasmonate (JA)-inducible proteins have been identified by microarray and proteomic analyses of insect-infested plants (Reymond et al., 2000; Collins et al., 2010). However, it has been shown that several plant defense proteins can successfully survive digestion in the insect gut and be eliminated in the frass, whereas proteins that do not have defensive functions, such as Rubisco, are rapidly degraded and not detected in frass. For example, it has been demonstrated that several jasmonate-inducible proteins (JIPs) actually retain enzymatic activity in the insect midgut and can be deleterious to the herbivore in the digestive tract (Chen et al., 2005). This finding implies that proteomic analysis of frass protein components is a novel approach for identifying plant proteins that are resistant to digestion and potentially function in herbivore defense (Chen et al., 2007). Using this technique, several JIPs were found in Manduca sexta frass that reduced the insect's ability to obtain essential nutrients from the plant (Chen et al., 2007). One of these proteins was a threonine deaminase isoform 2 (TD2; Chen et al., 2007). TD2 is synthesized as proenzyme, and when insects ingest tomato leaves, TD2 is converted to the processed form in their guts, where it reduces the concentrations of threonine, an essential amino acid for phytophagous insects (Chen et al., 2007). In this study, we used proteomic analysis of fall armyworm (FAW, Spodoptera frugiperda) frass to determine if there are possible herbivore-induced defensive proteins in maize that resist digestion. One of the predominant proteins found in this analysis was identified as ribosome-inactivating protein 2 (RIP2).
Ribosome-inactivating proteins (RIPs) are enzymes that have site-specific RNA N-glycosidase activity that arrest translation (Bass et al., 2004). They block translational elongation by depurinating residues on the large ribosomal RNA component (Endo & Tsurugi, 1987; Endo et al., 1987; Nielsen & Boston, 2001). In 1925, it was reported that pokeweed RIPs inhibit viral infection (Duggar & Armstrong, 1925; Irvin, 1983; Nielsen & Boston, 2001). Since then it has been shown that some RIPs function in defense against viruses, pathogens, and insects (Nielsen et al., 2001; Peumans et al., 2001; Bertholdo-Vargas et al., 2009). Based on their protein structure, RIPs are classified into three types (Nielsen & Boston, 2001). Type 1 RIPs consist of a single polypeptide chain with an approximate molecular mass of 30 kDa (Nielsen & Boston, 2001). Pokeweed antiviral protein, soapwort saporin, and barley translation inhibitor are type 1 RIPs (Nielsen & Boston, 2001). Type 2 RIPs have two polypeptide subunits that have an enzymatic domain and galactose-binding domain linked by disulfide bonds with an approximate molecular mass of 60 kDa (Nielsen & Boston, 2001). Ricin and abrin are well-known, highly toxic type 2 RIPs (Nielsen & Boston, 2001). Type 3 RIPs are synthesized as inactive precursors (proRIPs) that require proteolytic modification to form processed RIP (Nielsen & Boston, 2001). This processing step requires removal of c. 25 amino acids from the middle of the protein precursor coupled with additional processing at the N- and C-termini (Nielsen & Boston, 2001). Barley JIP60 and two maize RIP proteins belong to type 3 RIPs (Nielsen & Boston, 2001). The precursor form of the RIP1 protein is c. 32 kDa and after processing it comprises 16.5 and 8.5 kDa polypeptides that associate as a heterodimer (Bass et al., 2004). Unprocessed RIP2 protein is c. 30 kDa and the size of the processed subunits has not yet been reported.
There are several other important differences between the two RIP protein isoforms, RIP1 and RIP2, identified in maize (Bass et al., 2004). First, the amino acid sequences of RIP1 and RIP2 proteins are c. 73% similar (Bass et al., 1995). Secondly, the RIP1 protein is expressed in kernel, where it is believed to protect the seed from pathogen infection (Nielsen et al., 2001), whereas the RIP2 protein is expressed throughout the plant from the leaves to the tassel, but not the kernel (Bass et al., 2004). Thirdly, the RIP1 gene maps on chromosome 8 (bin 8.05) of the maize genome, while the RIP2 gene is located on chromosome 7 (bin 7.04), where there is a strong quantitative trait locus for caterpillar resistance (Bass et al., 1995, 2004; Brooks et al., 2005). These studies suggest that the RIP2 protein may play an important defensive role in vegetative tissues, as the RIP1 protein does in the kernel, but this has not been confirmed by directly testing the toxicity of RIP2 on insect herbivores.
The first objective of this study was to determine if putative herbivore defense proteins expressed in maize could survive digestion in the FAW gut and be excreted in the frass as was the case for M. sexta fed on tomato (Chen et al., 2005). The second objective was to determine if one of the putative defensive proteins, RIP2, identified in frass was induced by caterpillar feeding and toxic to FAW larvae.
- Top of page
- Materials and Methods
- Supporting Information
Plants have evolved with a number of defense mechanisms to protect themselves against insect herbivory. Because herbivores consume foliage that is used for growth and development, plants respond to herbivory by synthesizing a number of antinutritional substances (Berenbaum, 1995; Felton & Gatehouse, 1996; Felton, 2005; Zhu-Salzman et al., 2008). When insects ingest this cocktail of antinutritional proteins, it causes ‘indigestion’, limits their ability to fully utilize plant nutrients and impairs their growth (Felton, 2005). Studies have shown that some ingested plant defensive proteins remain intact in the insect gut and are eliminated in the frass (Chen et al., 2005, 2007; Jeffers et al., 2005; Zhu-Salzman et al., 2008).
Some of the maize proteins identified in FAW frass in this study have been implicated in plant defense against herbivores and pathogens. Beta-D-glucosidase has been shown to activate DIMBOA, a secondary metabolite toxic to the European corn borer (Ostrinia nubilalis; Yu et al., 2009). Lipoxygenase (LOX) and allene oxide synthase (AOS) catalyze steps in the JA biosynthetic pathway and the production of JA in response to herbivory triggers many plant defenses against insects (Howe & Jander, 2008). These results are supported by a previous study demonstrating that transcripts for two maize LOX genes (ZmLOX1 and ZmLOX3) and AOS increased in abundance when Mp708 and Tx601 plants are subjected to FAW feeding (Shivaji et al., 2010). In addition, maize 9-LOX (ZmLOX3) plays a defensive role against nematodes (Meloidogyne incognita) and fungi (Aspergillus flavus and Aspergillus nidulan; Gao et al., 2008, 2009). A putative fruit protein with unknown function that shares similarity with a pathogen-responsive oxidoreductase (drd-1) in potato (Montesano et al., 2003) was also present in the frass proteome. Another protein was an endo-beta-glucanase. In rice, an isoform of this enzyme responded to wounding, MeJA, and ethephon (Akiyama et al., 2009), which suggests its possible function in herbivore defense. Another protein that was identified was peroxidase. In maize, peroxidases have been shown to be associated with disease and caterpillar resistance (Dowd & Johnson, 2005; Dowd et al., 2010). The growth rates of two major maize pests (Helicoverpa zea and Lasioderma serricorne) were retarded when they were fed the maize peroxidase px11 (Chen et al., 2008). In addition to these proteins, a GDSL-like lipase (GLIP1) present in the frass proteome is similar to one in Arabidopsis that is involved in defense against the necrotrophic fungus Alternaria brassicicola (Oh et al., 2005). GLIP1, which is regulated by ET, triggers systemic resistance signaling in plants after fungal infection (Oh et al., 2005). Another Arabidopsis GDSL-like lipase (GLIP2) plays a role in plant defense against pathogens (Lee et al., 2009). Chitinases, which also were found in frass, have been shown to be defensive proteins in plants. For example, the overexpression of poplar chitinase (WIN6) in tomato retarded the development of Colorado potato beetle (Lawrence & Novak, 2006) and ectopic expression of a rice chitinase in peanuts enhanced resistance to Cercospora arachidicola (Iqbal et al., 2012). In addition, transcripts of the chitinase gene Prm3 have been shown to increase in response to FAW feeding in maize (Shivaji et al., 2010). One possible function of chitinases is to catalyze the disruption of the chitin-rich peritrophic matrix of the attacking herbivore. We also identified several peptides of leucine aminopeptidase in frass (Table 1). Tomato leucine aminopeptidase A (LapA) increases in response to wounding, exogenous MeJA, pathogen infection, and insect feeding (Chao et al., 1999; Chen et al., 2005, 2007; Zhu-Salzman et al., 2008). LapA protein has been detected in the midgut and frass of M. sexta (Chen et al., 2005, 2007), and its overexpression in tomato delays M. sexta growth and development (Lee et al., 2009). These proteomic studies indicated that a rich cocktail of putative maize herbivore defense proteins was present in FAW frass.
RIP genes are ubiquitous in the plant kingdom and more than 130 RIP genes have been identified (Girbes et al., 2004; Jiang et al., 2008). Expression and accumulation of these RIP-like proteins is regulated by a plethora of developmental, abiotic and biotic factors (Nielsen & Boston, 2001). In maize, the kernel isoform of the RIP1 protein has been extensively studied (Bass et al., 1992) and transgenic plants overexpressing this gene have been shown to inhibit insect growth (Dowd et al., 2003). In a more recent study, Dowd et al. (2012) reported that maize transformed with a single construct containing the coding sequences for the RIP1 protein, wheat germ agglutinin and tobacco hornworm chitinase inhibited the growth of H. zea and FAW larvae. But this construct is clearly more complicated than the native RIP2 gene sequence. Because the RIP1 protein is only expressed in the kernel, it is unlikely that it defends maize against leaf-feeding herbivores. The RIP2 protein, on the other hand, is induced by FAW feeding and inhibits growth of the herbivore. In addition, RIP1 and RIP2 share only 73% protein sequence similarity (Bass et al., 1995), so it is possible that these differences could result in differential degrees of toxicity between the two proteins.
Here we demonstrate that both the RIP2 protein and its transcripts are induced by FAW feeding in the whorl. Furthermore, we have shown that mechanically wounded plants treated with ET and JA, phytohormones known to be involved in signal transduction pathways leading to caterpillar defenses in maize (Harfouche et al., 2006; Ankala et al., 2009; Shivaji et al., 2010), expressed enhanced levels of RIP2. These results, in conjunction with its indigestibility, strongly suggested that it could be involved in herbivore defense. To test this, FAW larvae were fed both unprocessed and processed recombinant RIP2 protein in a physiologically relevant concentration. The results indicated that both forms inhibited FAW growth by c. 25%. In addition, peptide fragments of the recombinant RIP2 protein detected in the frass were similar in size to those present in frass from caterpillars that fed on maize. Only a few studies have investigated the mechanism of RIP protein toxicity to insects. SNA-I from elderberry caused cell apoptosis in the gut tissues of Acyrthosiphon pisum and Spodoptera exigua (Shahidi-Noghabi et al., 2010b). SNA-I and SNA-II also induced caspase-3 like activity in the midgut cell line of Lepidoptera (Shahidi-Noghabi et al., 2010a). These findings suggest that RIP proteins are toxic to insect herbivores because they trigger apoptosis in the midgut; however, the mechanism of RIP2 protein action on the FAW midgut has not yet been investigated.
The rapid induction of RIP2 transcripts and RIP2 protein in response to caterpillar feeding could be an early deterrent against insect herbivory. In the caterpillar-resistant maize inbred Mp708, another defensive enzyme, the cysteine protease Mir1-CP, accumulates within 1 h after insect attack (Pechan et al., 2000). Other genes that are rapidly induced in response to wounding in maize include wound-induced proteinase inhibitor (WIP1), as early as 30 min after wounding (Rohrmeier & Lehle, 1993), and maize proteinase inhibitor (MPI) at 20 min after wounding (Tamayo et al., 2000). Therefore, it appears that herbivore feeding rapidly activates a suite of defensive proteins in maize.
In addition to being rapidly induced, the RIP2 protein persists in the leaves up to four d. One explanation for its long half-life is that it responds to the insect feeding behavior. Caterpillars do not continuously feed on plants and eating bouts are often separated by multiple gaps (Reynolds et al., 1986). During these gaps, they need time to digest leaf tissue or prepare to molt. Because molting in the field usually takes 1 or 2 d, plants need to maintain their defenses for the next insect attack; therefore the RIP2 protein could be deployed for long-term defense. In addition, it could protect the plant from the occurrence of fungal infection in the open wound sites caused by herbivory. Because the kernel RIP1 protein inhibits fungal growth, it is possible that the foliar RIP2 protein might function in a similar manner (Nielsen et al., 2001).
We also observed that the size of unprocessed RIP2 proteins varied among the inbred lines tested (Fig. S2). However, a comparison of the cDNA sequences of RIP2 from Mp708, Tx601 and B73 indicated that the derived amino acid sequences were nearly identical. So, it seems unlikely that minor changes in the amino acid sequence would significantly alter their size. Therefore, size differences could be due to some type of post-translational modification.
As mentioned previously, RIP1 and RIP2 map to different physical locations on the maize genome. RIP2 is on chromosome 7 whereas RIP1 is located on chromosome 8 (Bass et al., 1995, 2004). Maize chromosomes 1, 5, 7, and 9 contain major loci for insect resistance to FAW and the southwestern corn borer (Diatraea grandiosella; Brooks et al., 2007). The RIP2 gene lies within strong quantitative trait locus for caterpillar resistance in maize (Brooks et al., 2005, 2007). This, in conjunction with its ability to impair FAW growth by c. 25%, suggests that the RIP2 protein is an important member of a group of herbivore-induced resistance proteins in maize.