Serendipita indica modulates expression of arginine metabolism genes during colonization of Arabidopsis thaliana roots

Endophytic fungus Serendipita indica belongs to the order Sebacinales. It is an extremely versatile root endophyte colonizing more than 150 different plant species including the model plant Arabidopsis thaliana. Colonization of S. indica triggers enhanced growth, early flowering, increase in seed content, alteration of secondary metabolite pathways and adaption to different biotic and abiotic stresses. This is why this fungus got ample attention by the plant researchers. In this study, we report modulation in expression of argininosuccinate synthase (AS), argininosuccinate lyase (AL) and arginase 2 (ARGAH2) genes in A. thaliana as a result of S. indica colonization. Similarly, expression of GUS reporter gene driven by AS and ARGAH2 promoters in roots was altered. AS, AL and ARGAH2 genes were down-regulated at early stage of root colonization i.e at 3 days post inoculation (dpi), with a subsequent up-regulation at later stages (7 and 14 dpi). Other arginine catabolism genes, Arginine decarboxylase-1 (ADC1), Arginine decarboxylase-2 (ADC2) and Pyrroline-5-carboxylate reductase (P5CR) were strongly upregulated at later stages of S. indica colonization. Our findings show significant changes in expression of several important genes in arginine metabolic pathway upon S. indica colonization. These changes are very specific and strongly depend on the colonization phase of the fungus.


Introduction
Abiotic and biotic stresses are the major causes of crop losses throughout the world.They primarily include unfavorable climate conditions, different plant diseases and plant pests.One commonly used solution to cope with these stresses is the high chemical input to increase the plant production [1].However, these soil and plants treatments cause severe damage to environment.Use of biological agents instead of chemical treatments is therefore gaining more attention and importance.Mycorrhizae are already established as beneficial associations of fungi with plants increasing their growth and production.Moreover, endophytic arbuscular mycorrhizal fungi (AMF) are proven to enhance abiotic stress tolerance.These fungi have mutualistic relationships with 80% plant species worldwide [2].In addition, many AMF associations were shown to be effective against soil born plant pathogens [3,4] and play an important role in nutrient acquisition of host plants [5].Endophytic fungus Serendipita indica (previously known as Piriformospora indica) belongs to the order Sebacinales.It colonizes roots, grows inter-and intracellularly, and forms spores in the roots as well as on the root surface.Its colonization on A. thaliana roots can be divided into four different phases: (1) extracellular (approx. 1 dpi); ( 2) biotrophic (less than 3 dpi); (3)  Homozygous T3 (Third generation) seeds of lines pAS::GUS [9] and pARGAH2::GUS [15] were collected after segregation analysis and used for final characterization of expression patterns.

Histochemical GUS assay
Histochemical GUS expression was performed using X-GLUC (5-bromo-4 chloro-3-indolyl β-D-glucuronic acid) (Biomol, Germany).Three, 7 and 14 days post inoculation (dpi) petri dishes containing inoculated plants were opened and X-GLUC solution was added.Dishes were then vacuum-infiltrated for 3 min and incubated at 37 °C for eight hours.After incubation, dishes were washed with 70 % ethanol and observed under dissection microscope for GUS staining.Photographs were taken with Axiovert 200M (Zeiss, Austria) using an Axiocam digital camera (Zeiss, Austria).

Sample collection, RNA isolation and quantitative RT-PCR
Root segments were harvested at 3, 7 and 14 dpi from colonized and uncolonized (control) plants of the same age omitting root tips.Samples were immediately shock-frozen in liquid nitrogen.Three plates were used for harvesting at each time point representing one biological replicate.Three biological replicates from independently grown batches were collected for RNA isolation.All experiments were repeated three times.RNA was extracted as described in [9].Quantitative RT-PCR was performed using an ABI PRISM 7300 Sequence Detector (Applied BioSystems; USA).The PCR was carried out at 50 °C for 2 min, 95 °C for 5 min followed by 40 cycles at 95 °C for 15 s, 60 °C for 30 s and 72 °C for 1 min.Primer sequences are listed in (Table 1).18S RNA primers were used as internal reference.Fold changes were calculated using 2 -ddCt method [16].RNA samples were used from three independent biological replicates.Each biological replicate was run in a set of three technical replicates to minimize the experimental errors.

Statistical analysis
Gene expression was calculated using 2 -ddCt method.Statistical variations were tested using ANOVA and LSD tests (p < 0.05) using the statistical software package STATGRAPHICS Plus Version 5.0.

Characterization of promoter::GUS transgenic lines
Temporal expression patterns of previously published pAS::GUS and pARGAH2::GUS fusion lines were analyzed [9, 15].Here, the promoter activity of AS and ARGAH2 was studied in S. indica-colonized roots of A. thaliana.pAS::GUS lines showed very weak promoter activity at 3 dpi (Figure 1A).In case of pARGAH2::GUS line promoter activity showed the same trend at 3 dpi (Figure 1D).
In contrast, at 7 dpi pAS::GUS activity was observed only in vascular bundles (Figure 1B), and in pARGAH2::GUS line GUS staining could be observed scattered all over the roots (Figure 1E).At 14 dpi in pAS::GUS line, strong GUS activity was found in most regions of the analyzed root tissue (Figure 1C).In case of pARGAH2::GUS line strong promoter activity was also found all over the roots (Figure 1F).These findings were confirmed by at least two independent transgenic lines for both of the genes with consistent and reproducible results.

Expression analysis of arginine cycle genes
Important genes for two anabolic and three catabolic enzymes of arginine metabolic pathway were selected for expression analysis.The entire arginine pathway is presented in (Figure 2).Expression of these genes was analyzed using qRT-PCR at three different time points: early 3 dpi, mid 7 dpi and late 14 dpi.At an early stage of development (3 dpi), argininosuccinate synthase (AS) showed down-regulation as compared to un-colonized control roots, with a subsequent up-regulation at later time points.However, gene expression was decreased at 14 dpi as compared to 7 dpi.Similarly, argininosuccinate lyase (AL) showed the same trend i.e down-regulation at 3dpi and up-regulation at later time points.But in this case gene expression was not as strong as in case of AS (Figure 3A & B).In case of arginase related genes, arginase 1 (ARGAH1) gene was found up-regulated at all three time points with a strong upregulation at 7dpi followed by a decreasing trend.Arginase 2 (ARGAH2) showed a significant down-regulation at 3dpi with almost two-fold up-regulation at later time points (Figure 3C & D).Among other catabolic genes, i.e.ADC1, ADC2 and P5CR, an increasing trend was found at all-time points tested.Strongest up-regulation was found at 14 dpi in all three genes (Figure 3E, F & G).

12, 13].
Presence of the promoter was confirmed by sequencing of the resulted plasmid.Floral dip technique [14] was used for Agrobacterium-mediated plant transformation to insert the binary vectors in A. thaliana (Col-0).Screening of transformed seeds was done by antibiotic selection.

42]. Conclusion This
[38]hown here, the gene encoding this enzyme is up-regulated at 7 and 14 dpi.Increased proline production can be beneficial for many plant responses.For instance, it is involved in osmotic stress management and accumulates as important osmolyte during stress conditions[38]and as a result of dehydration [39].Accumulation of proline can be a result of incompatible plant microbe interactions [40] and can enhance the root growth by increasing the root meristematic zone [41].Similarly, AMF are known to be triggering increased root growth [study demonstrates that S. indica triggers sophisticated modulations of the arginine pathway in colonized A. thaliana roots on the gene expression level.Genes encoding key enzymes in this pathway such as AS, AL and ARGAH2 show significant down-regulation at early time points, whereas during later stages they are strongly and significantly up-regulated.These alterations are tightly interconnected with specific colonization phases of the fungus and are most probably indispensable for successful colonization and further development of the mutualistic relationship.Controlled arginine metabolism might not only help S. indica to avoid initial plant defense responses but also might improve conditions to sustain its further growth.Whether S. indica does this either to cope with NO-mediated plant defenses or to synthesize important metabolites essential for its growth and development should be in focus of further studies.Authors' contributions Conceived and designed the experiments: S Anwar & K Wieczorek, Performed the experiments: S Anwar, Analysed the data: S Anwar, Contributed materials/ analysis/ tools: K Wieczorek, Wrote the paper: S Anwar & K Wieczorek.