Glycan-binding F-box protein from Arabidopsis thaliana protects plants from Pseudomonas syringae infection.

BACKGROUND: A small group of F-box proteins consisting of a conserved F-box domain linked to a domain homologous to the glycan-binding protein has been identified within the genome of Arabidopsis thaliana. Previously, the so-called F-box-Nictaba protein, encoded by the gene At2g02360, was shown to be a functional lectin which binds N-acetyllactosamine structures. Here, we present a detailed qRT-PCR expression analysis of F-box-Nictaba in Arabidopsis plants upon different stresses and hormone treatments. RESULTS: Expression of the F-box-Nictaba gene was enhanced after plant treatment with salicylic acid and after plant infection with the virulent Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000). ß-glucuronidase histochemical staining of transgenic Arabidopsis plants displayed preferential activity of the At2g02360 promoter in trichomes present on young rosette leaves. qRT-PCR analyses confirmed high expression of F-box-Nictaba in leaf trichomes. A. thaliana plants overexpressing the gene showed less disease symptoms after Pst DC3000 infection with reduced bacterial colonization compared to infected wild type and F-box-Nictaba knock-out plants. CONCLUSIONS: Our data show that the Arabidopsis F-box-Nictaba gene is a stress-inducible gene responsive to SA, bacterial infection and heat stress, and is involved in salicylic acid related plant defense responses. This knowledge enriched our understanding of the physiological importance of F-box-Nictaba, and can be used to create plants with better performance in changing environmental conditions.

Transcriptional behavior of EUL-related rice lectins toward important abiotic and biotic stresses.

The rice genome encodes several genes for putative carbohydrate-binding proteins belonging to the family of Euonymus related lectins (EULs). This lectin family was discovered recently and evidence shows that the expression of these proteins is subject to multiple environmental stresses. In this study, quantitative reverse transcription PCR (qRT-PCR) was conducted on rice seedlings exposed to various abiotic (150mM NaCl, 100mM mannitol, and 100µM abscisic acid (ABA)) and biotic (Xanthomonas oryzae pv. oryzae and Magnaporthe oryzae) stresses to compare the transcriptional behavior of the EULs and a known stress related lectin Orysata belonging to the family of jacalin-related lectins. All EUL transcripts were strongly up-regulated after ABA and NaCl treatments in the roots whereas the overall expression level was generally lower and more variable in the shoots. Moreover, all abiotic stresses induced Orysata in both tissues except for mannitol treatment which failed to show an effect in the roots. Orysata also strongly accumulated after X. oryzae pv. oryzae infection, as were various D-type EUL lectins. In contrast, some of the EUL proteins, including OrysaEULS3, OrysaEULD1A and OrysaEULD2, as well as Orysata were significantly down-regulated upon M. oryzae attack, suggesting fungal manipulation of these genes. Collectively, our results clearly show that rice expresses multiple carbohydrate-binding proteins in response to a wide variety of abiotic and biotic stress conditions. We hypothesize that the Euonymus related proteins fulfill a prominent role in sensing and responding to multiple environmental cues.

Orysata, a jacalin-related lectin from rice, could protect plants against biting-chewing and piercing-sucking insects.

The present study reports the insecticidal activity of Orysata, a lectin from rice with mannose specificity, belonging to the family of jacalin-related lectins. The effect of Orysata was investigated against three important pest insects in agriculture: the beet armyworm Spodoptera exigua Hübner (Lepidoptera: Noctuidae), and two aphid pests: green peach aphid Myzus persicae Sulzer and pea aphid Acyrthosiphon pisum (Hemiptera: Aphidoidea). Bioassays with S. exigua and M. persicae were performed using detached leaves from transgenic tobacco lines overexpressing Orysata. The expression levels ranged between 38 and 71 µg/g FW, corresponding to 0.6-1.1% of total soluble protein. Intoxicated larval stages of S. exigua revealed significant mortality, reductions in larval weight gain and a retardation of development. Similarly, feeding on leaves expressing Orysata lowered the mortality of the green peach aphids significantly. When pea aphids were fed on an artificial diet supplemented with different amounts of recombinant Orysata, mortality was high at relatively low lectin concentrations; the estimated 50% lethal concentration being 79 µg/ml. In conclusion, our results demonstrated that the jacalin-related lectin Orysata possesses strong insecticidal activity, suggesting that it can be considered as a valuable candidate to be used as a control agent against both biting-chewing and piercing-sucking pest insects.

Characterization of a type D1A EUL-related lectin from rice expressed in Pichia pastoris.

OrysaEULD1A is one of the five EUL genes in rice (Oryza sativa) encoding a putative carbohydrate-binding protein belonging to the family of Euonymus related lectins (EUL). The OrysaEULD1A sequence comprises two highly similar EUL domains (91% sequence similarity and 72% sequence identity) separated by a 23 amino acid linker sequence and preceded by a 19 amino acid N-terminal sequence. In the present study, the full-length protein OrysaEULD1A as well as its individual domains OrysaEULD1A domain 1 and 2 were expressed in Pichia pastoris. After purification of the recombinant proteins, their carbohydrate-binding specificity was analyzed and compared. Interestingly, all recombinant lectins showed clear specificity towards galactosylated structures. Furthermore, all recombinant proteins agglutinated red blood cells, indicating that the full-length protein OrysaEULD1A and its domains are true lectins. These results taken together with data previously reported for single-domain EUL proteins indicate that although the amino acids--responsible for the formation of the carbohydrate-binding site--are identical for all EUL proteins in rice, these lectins show different carbohydrate specificities. This promiscuity of the carbohydrate-binding site can be attributed to gene divergence.

Expression analysis of a type S2 EUL-related lectin from rice in Pichia pastoris.

Rice (Oryza sativa) expresses different putative carbohydrate-binding proteins belonging to the class of lectins containing an Euonymus lectin (EUL)-related domain, one of them being OrysaEULS2. The OrysaEULS2 sequence consists of a 56 amino acid N-terminal domain followed by the EUL sequence. In this paper the original sequence of the EUL domain of OrysaEULS2 and some mutant forms have been expressed in Pichia pastoris. Subsequently, the recombinant proteins were purified and their carbohydrate binding properties determined. Analysis of the original protein on the glycan array revealed interaction with mannose containing structures and to a lesser extent with glycans containing lactosamine related structures. It was shown that mutation of tryptophan residue 134 into leucine resulted in an almost complete loss of carbohydrate binding activity of OrysaEULS2. Our results show that the EUL domain in OrysaEULS2 interacts with glycan structures, and hence can be considered as a lectin. However, the binding of the protein with the array is much weaker than that of other EUL-related lectins. Furthermore, our results indicate that gene divergence within the family of EUL-related lectins lead to changes in carbohydrate binding specificity.

Expression analysis of the nucleocytoplasmic lectin 'Orysata' from rice in Pichia pastoris.

The Oryza sativa lectin, abbreviated Orysata, is a mannose-specific, jacalin-related lectin expressed in rice plants after exposure to certain stress conditions. Expression of a fusion construct containing the rice lectin sequence linked to enhanced green fluorescent protein in Bright Yellow 2 tobacco cells revealed that Orysata is located in the nucleus and the cytoplasm of the plant cell, indicating that it belongs to the class of nucleocytoplasmic jacalin-related lectins. Since the expression level of Orysata in rice tissues is very low the lectin was expressed in the methylotrophic yeast Pichia pastoris with the Saccharomyces α-factor sequence to direct the recombinant protein into the secretory pathway and express the protein into the medium. Approximately 12 mg of recombinant lectin was purified per liter medium. SDS/PAGE and western blot analysis showed that the recombinant lectin exists in two molecular forms. Far western blot analysis revealed that the 23 kDa lectin polypeptide contains an N-glycan which is absent in the 18.5 kDa polypeptide. Characterization of the glycans present in the recombinant Orysata revealed high-mannose structures, Man9-11 glycans being the most abundant. Glycan array analysis showed that Orysata interacts with high-mannose as well as with more complex N-glycan structures. Orysata has potent anti-human immunodeficiency virus and anti-respiratory syncytial virus activity in cell culture compared with other jacalin-related lectins.