An efficient method for visualization and growth of fluorescent Xanthomonas oryzae pv. oryzae in planta.(Methodology article)(Report)

Authors: Sang-Wook Han (equal contributor) [1]; Chang-Jin Park (equal contributor) [1]; Sang-Won Lee (equal contributor) [1]; Pamela C Ronald (corresponding author) [1]

Background

Xanthomonas oryzae pv. oryzae , a yellow-pigmented Gram-negative bacterium, is the causal agent of bacterial blight disease of rice (Oryzae sativa L.) plants. X. oryzae pv. oryzae infection can cause yield loss of up to 50% in tropical Asia [1]. X . oryzae pv. oryzae infects rice leaves through natural openings such as hydathodes and/or wounded sites and then primarily colonizes the vascular tissues by propagating in the xylem. Increased extracellular polysaccharide secretion accompanies bacteria growth, eventually causing a block in the vascular system [2]. The early symptoms therefore start with wilting in the infected leaves and enlargement in length and width of the legions of leaf blight [3]. As the symptom progress, severe necrosis occurs along the interveinal regions. Eventually, the whole leaf becomes whitish and greyish, and then dies [2, 4]. Over the last few decades, the challenge in elucidating biological phenomena has been met by advances in techniques, which have accelerated our understanding of biological events. In particular, useful tools have been developed to evaluate cellular dynamics in vivo . One of the best examples of a technique that has facilitated cell-based studies is marker systems that use fluorescent proteins (FPs). Since the green fluorescent protein (GFP) was first discovered from the jellyfish Aequorea victoria in 1962 [5], various types of FPs, including red, yellow, and cyanine fluorescent proteins, have been developed and used in fields such as biophysics, biochemistry, and plant pathology [6, 7, 8]. The proteins are stable [9], non-species specific [10, 11], and have no requirement of specific substrate [11]. Therefore, labelling a specific target protein with a FP is a powerful molecular tool for a cell biology study since it provides the ability to visualize, track, and quantify targets in living cells with high spatial and temporal resolution essential features for understanding biology systems [8]. In addition, bimolecular fluorescence complementation (BiFC) analysis using split-FP systems has been successfully applied to determine protein-protein interactions in planta as well as in animals [12, 13]. Recently, FPs have been used for monitoring living organisms such as Lactobacillus sakie , Pseudomonas syringae , X. axonopodis pv. dieffenbachiae , and Xylella fastidiosa in their hosts [14, 15, 16, 17].

To determine virulence and pathogenicity of bacterial pathogens in plant-microbe interactions, researchers typically quantify bacterial multiplication

in planta by counting the number of bacterial colonies in plate assays of leaf extracts [18, 19]. This method consists of leaf harvesting, tissue maceration, colony plating, incubating, and counting of bacterial cells. These methods are time-consuming and labour intensive, especially for slow-growing bacteria like X. oryzae pv. oryzae . In addition, the results frequently show large amounts of variation depending on experimental conditions.

Here, we provide an improved method for the study of

X. oryzae pv. oryzae using a fluorescent marker system. It not only eliminates many of the difficulties of conventional methods but also allows for reliable and rapid monitoring of bacteria in planta prior to the formation of symptoms.

Results and discussion

Generation of

X. oryzae pv. oryzae expressing GFPX. oryzae pv. oryzae Philippine race 6 (PXO99) carrying the gfp gene was generated using pPneo -gfp , which is a new construct based on the broad-host-range probe vector, pPROBE-gfp [20] and the pML122 vector [21]. The pPROBE-gfp plasmid was previously used as a marker to assess expression of genes of interest in Xyllela festidosa and X. campestris pv. campestris [22]. The neomycin promoter in pML122 was successfully used for protein expression in X. oryzae pv. oryzae [23]. To generate a plasmid, pPneo -gfp , which is approximately 400 nucleotides long and contains the neomycin promoter, was taken from the pML122 plasmid and ligated into a pPROBE-gfp vector. The modified construct was introduced into PXO99 wild type strain and transformants were selected on peptone sucrose agar (PSA) plate containing kanamycin (50 [mu]g/ml) for selection of pPneo -gfp . Expression of GFP in the transformants was tested by Western-blot analysis using an anti-GFP antibody (data not shown). X. oryzae pv. oryzae carrying pPneo -gfp (PXO99GFP ) showed strong fluorescence (Fig. 1). Our results demonstrate that the PXO99GFP strain carrying the pPneo -gfp vector constitutively and strongly expresses GFP in X. oryzae pv. oryzae , suggesting that the marker system might be applied to study plant-microbe interactions in planta .Figure 1: Observation of Xanthomonas oryzae pv. oryzae carrying green fluorescence protein . PXO99GFP selected on PSA plates containing …