Characterization of a Beta Vulgaris Polygalacturonase- Inhibiting Protein: a Defense Response Gene

Polygalacturonase-inhibiting proteins (PGIPs) are plant cell wall proteins that inhibit pathogen and pest polygalacturonases (PGs). PGIPs are members of the leucine-rich repeat (LRR) protein family that play crucial roles in development, pathogen defense and recognition of beneficial microbes in plants. Two sugar beet PGIP genes, Bv(FC607)PGIP1 and Bv(FC607)PGIP2, were cloned from a breeding line FC607. Sequence analysis showed that both genes encoded about 380 amino acids and shared 74.8% sequence similarity. They were most closely matched to PGIPs of a sugar beet line KWS2320 and a subgroup of M. truncatula PGIP (GenBank No: XP_003621816). FCPGIPs exhibited characteristics of other plant PGIPs, including the presence of an N-terminal signal peptide and LRR repeats that were50-60 amino acid longer than has been reported for most other PGIPs. In 2-month old plants, RT-PCR analysis demonstrated that each PGIP gene was expressed constitutively, with maximum expression being observed in roots, followed by leaves then petioles and hypocotyls, suggesting that the gene is developmentally regulated. A study of PGIP inhibitory effect on pathogens and pests is ongoing. Introduction Polygalacturonase-inhibiting proteins (PGIPs), produced in most plant cell walls, effectively and specifically combine with pathogen and pest polygalacturonases (PGs) and inhibit their activity used for overcoming plant defense responses (D’Ovidio et al., 2004). Phytopathogenic fungi, bacteria, nematodes and insects are known to secrete PG enzymes for breaking down the polygalacturonate chain in plant cell walls. The interaction between PGs and plant PGIPs favors the accumulation of oligogalacturonides which elicit a wide range of plant defense responses (Gomathi and Gnanamanickam 2004; Schacht et al., 2011). Many plants possess more than one PGIP with differential abilities to inhibit different PGs of pathogens. Like the products of many resistance genes, PGIPs belong to the subclass of proteins containing leucine-rich repeats (LRRs) of the extracytoplasmic type (Jones and Jones 1997). In our laboratory, one of the sugar beet genes that codes for PGIP has been found to be induced by the sugar beet root maggot in roots (Puthoff and Smigocki, 2007). In order to characterize the structure and functional features of sugar beet PGIPs, several PGIP genes were cloned and characterized from several sugar beet breeding lines. Here we report on the cloning of two PGIP genes from sugar beet line FC607 and on their tissue-specific expression in 2-month old plants. These findings will facilitate further studies on PGIP inhibition of pathogens and pests and will advance the development of novel approaches for more effective disease and pest control in sugar beet. Materials and Methods Plant materials A sugar beet breeding line FC607 was used in this study. Seeds were germinated at room temperature in Pro-Mix (Professional Horticulture) soil. Seedlings were grown in the growth chamber at 24°C with a 16 h photoperiod. Full-length cDNA cloning and sequence analysis The full-length sequence of the sugar beet PGIP cDNA was obtained through rapid amplification of cDNA ends polymerase chain reaction (RACE PCR) using the SMARTer RACE cDNA Amplification Kit (Clontech, Mountain View, CA). Gene specific primers were designed and the RACE fragment was amplified as previously reported (Padmanaban et al., 2011). PCR amplified fragments were sequenced and nucleotide BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) searches were performed using default parameters. Amino acid sequences and opening reading frames were determined by ExPASy translation tool (http://web.expasy.org/translate/). Using genomic DNA and gene specific primers, PGIP genomic DNA was cloned by PCR and the resulting DNA fragment was sequenced. The SignalP 4.1 server (Petersen et al., 2011) was used to predict the signal peptide. The LRR domains were predicted according to sequence alignment of BvPGIPs and the bean PvPGIP2 whose secondary crystallographic structure has been determined (Di Matteo et al., 2003). Amino acid sequences of BvPGIPs and other PGIPs were aligned by ClustalW through MEGA6 program (www.megasoftware.net). Phylogenetic tree was constructed with the neighbor-joining method (Tamura et al., 2013). Analysis of PGIP gene expression Total RNA was extracted from leaves, petioles, hypocotyls, and roots of 2-month old plants using RNeasy Plant Mini Kit (Qiagen, Valencia, CA). RT-PCR was carried out on 100 ng of total RNA using Titanium One-Step RT-PCR Kit (Clontech) under the following conditions: 50oC for 1 h, 94oC for 2 min, 30 cycles of 94oC for 30 sec, 60oC for 30 sec, 72oC for 1 min, followed by 72oC for 5 min. RT-PCR results were normalized to transcripts of the constitutively expressed plant actin gene (Smigocki et al., 2013). Gene expression was quantified by densitometry with an AlphaImager HP (Alpha Innotech, San Leandro, CA). RT-PCR analyses were repeated three times. Results and Discussion Cloning and analysis of Beta vulgaris PGIPs To investigate intergenotype variation in sugar beet PGIP genes, the full complements of PGIP genes of the B. vulgaris breeding line FC607 were cloned and characterized. To date, two cDNA fragments were cloned from FC607 using primers based on the PGIP EST sequence (GenBank No: DV501910) isolated from sugar beet root after insect feeding (Puthoff and Smigocki, 2007). Comparison of the nucleotide and deduced amino acid sequences using BLAST revealed that the cloned cDNAs shared the highest sequence homology with other known PGIP genes. Comparison of Bv(FC607)PGIP1 and Bv(FC607)PGIP2 cDNAs and PCR derived genomic DNA sequences revealed that neither gene had introns. The full-length cDNA sequences of 1,152 and 1,146 bp, encoded 384 and 382 amino acids, respectively, with a calculated molecular weight of about 49.0 kDa. These two peptides showed 74.8% similarity, and shared the typical PGIP topology, which included a signal peptide for secretion, a 75-amino acid N-terminal domain, a domain comprising leucine rich repeats (LRRs), and a 21-amino acid C-terminal domain (Fig. 1). A total of eleven imperfect LRR regions of about 24 amino acids each was predicted according to sequence alignment of Bv(FC607)PGIPs with the bean PvPGIP2 whose secondary crystallographic structure has been determined (Di Matteo et al., 2003). However, according to the LRR search program (http://lrrsearch.com/), only 10 LRR domains (excludes the first one) were predicted for Bv(FC607)PGIP1 and only 9 LRRs (excludes the second and the ninth LRRs) were predicted for Bv(FC607)PGIP2. Nevertheless, the LRR region of these two genes exhibited the identical consensus sequence (LxxLxLxxNxLxGxIPxxLGxLxx) and matched precisely the extracytoplasmic LRR consensus sequence of other resistance genes (Jones and Jones 1997). There were 4 potential Nglycosylation sites (NxS/T; where x can be any amino acid), three of which occurred in the LRR domain, where the third NxS/T site was different between these two genes. Both genes contained 5 cysteine residues at the N-terminal and C-terminal regions. Sequence alignment by ClustalW revealed that both Bv(FC607)PGIP1 and Bv(FC607)PGIP2 showed the closest homology with PGIP or PGIP-like proteins from a B. vulgaris line KWS2320, a recently released genome sequence (Dohm et al., 2014). A 97.4% similarity between Bv(FC607)PGIP1 and Bv(KWS2320)PGIP (accession No: XP_010675711) and an 80.0% similarity between Bv(FC607)PGIP2 and Bv(KWS2320)PGIP (XP_010676004) was observed (Fig. 2A, 2B). The second closest homology of our sugarbeet PGIPs was to Medicago truncatula PGIP (MtPGIP2, XP_003621816), followed by Nicotiana tabacum PGIP (NtPGIP, AIA22327), and Arabidopsis thaliana PGIP (AtPGIP2, AAF69828). Like most of the PGIPs from B. vulgaris KWS2320, Bv(FC607)PGIP1 and Bv(FC607)PGIP2 were approximately 50 amino acids longer than the PGIP structure model PvPGIP2 that represents most of other known plant PGIPs (Fig. 2A). The additional amino acids were found in the N-terminal and the LRR regions of Bv(FC607)PGIP1 and Bv(FC607)PGIP2 LRR but not found in most other plant PGIPs, thus potentially being unique to the sugar beet PGIPs .