Abstract
Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) is a well-known pathogen and model organism used to study plant-pathogen interactions and subsequent plant immune responses. Numerous studies have demonstrated the effect of Pst DC3000 on Arabidopsis plants and how type III effectors are required to promote bacterial virulence and pathogenesis. F-Box Nictaba (encoded by At2g02360) is a stress-inducible lectin that is upregulated in Arabidopsis thaliana leaves after Pst DC3000 infection. In this study, a flood inoculation assay was optimized to check the performance of transgenic Arabidopsis seedlings with different expression levels of F-Box Nictaba after bacterial infection. Using a combination of multispectral and fluorescent imaging combined with molecular techniques, disease symptoms, transcript levels for F-Box Nictaba, and disease-related genes were studied in Arabidopsis leaves infected with two virulent strains: Pst DC3000 and its mutant strain, deficient in flagellin ΔfliC. Analyses of plants infected with fluorescently labeled Pst DC3000 allowed us to study the differences in bacterial colonization between plant lines. Overexpression plants showed a reduced bacterial content during the later stages of the infection. Our results show that overexpression of F-Box Nictaba resulted in reduced leaf damage after bacterial infections, whereas knockdown and knockout lines were not more susceptible to Pseudomonas infection than wild-type plants. In contrast to wild-type and knockout plants, overexpressing lines for F-Box Nictaba revealed a significant increase in anthocyanin content, better efficiency of photosystem II (Fv/Fm), and higher chlorophyll content after Pst DC3000 infection. Overexpression of F-Box Nictaba coincided with increased expression of salicylic acid (SA) related defense genes, confirming earlier data that showed that F-Box Nictaba is part of the SA-dependent defense against Pst DC3000 infection. Knockout lines yielded no discernible effects on plant symptoms after Pseudomonas infection suggesting possible gene redundancy between F-Box Nictaba genes.
Highlights
Lectins are defined as proteins that selectively recognize and reversibly bind to specific carbohydrate structures without making structural changes to the carbohydrate moiety
To explore the role of F-Box Nictaba (At2g02360) in the plant defense of Arabidopsis thaliana against bacterial infection, 2week-old plants were infected with Pseudomonas syringae using the flood inoculation technique (Ishiga et al, 2011)
Transcript levels for At2g02360 were 2-fold upregulated in 2-week-old wildtype Arabidopsis plants at 3 days after Pst DC3000 infection. These data are similar to the results described for 5-week-old Arabidopsis plants infected by spraying with a bacterial solution of Pst DC3000 (Stefanowicz et al, 2016)
Summary
Lectins are defined as proteins that selectively recognize and reversibly bind to specific carbohydrate structures without making structural changes to the carbohydrate moiety These carbohydrate-binding proteins are present in all kingdoms of life and have been studied extensively in animals and plants, and occur in bacteria, fungi, and viruses (Peumans and Van Damme, 1995; Lannoo and Van Damme, 2010; Van Holle and Van Damme, 2018). The classical lectins are constitutively expressed and mainly reside in the vacuole, in contrast to the stress-inducible lectins located in the nucleus and the cytoplasm The latter group of lectins is expressed at a basal level under normal growth conditions, but their expression is upregulated when plants face unfavorable conditions, such as biotic or abiotic stresses (Van Holle and Van Damme, 2018). Understanding the protein-carbohydrate interactions in plant cells is crucial to discern the mode of action of lectins (Lannoo and Van Damme, 2010)
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