Role of Sulphate Transporter (PiSulT) of Endophytic Fungus … · 2020. 1. 8. · 1 1 Role of Sulphate Transporter (PiSulT) of Endophytic Fungus Serendipita indica in Plant 2 Growth

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Role of Sulphate Transporter (PiSulT) of Endophytic Fungus Serendipita indica in Plant 1

Growth and Development 2

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Om Prakash Narayan1, Nidhi Verma1, Abhimanyu Jogawat1, Meenakshi Dua2 and Atul 4

Kumar Johri1* 5

6 1School of Life Sciences, Jawaharlal Nehru University, New Mehrauli Road, New Delhi-110067, 7

India. 8

9 2School of Environmental Sciences, Jawaharlal Nehru University, New Mehrauli Road, New 10

Delhi-110067, India. 11

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*Corresponding author Email: [email protected] 13

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Keywords: Serendipita indica, sulphate transporter, maize, colonization, PiSulT 15

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Short title: PiSulT regulates plant growth 17

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One-Sentence Summary: High-affinity sulphate transporter of Serendipita indica (PiSulT) 19

transfer sulphate from soil to plant under low sulphate condition and improve plant growth and 20

development. 21

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The authors responsible for the distribution of materials integral to the findings presented in this 23

article in accordance with the policy described in the instructions for authors (www.plantcell.org) 24

is Atul Kumar Johri ([email protected]). 25

26

ABSTRACT 27

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Sulfur is an important macronutrient required for the growth, development of plants and is 29

a key component of many metabolic pathways. We have functionally characterized a high-affinity 30

sulphate transporter (PiSulT) from an endophytic fungus Serendipita indica. The PiSulT belongs to 31

the major facilitator superfamily (MFS) of membrane transporter. The PiSulT functionally 32

complements the yeast sulphate transporter mutant HK14. PiSulT is a high-affinity sulphate 33

transporter, having Km 15µM. We found enhanced expression of PiSulT in external fungal hyphae 34

which helps the fungus in the acquisition of sulphate from the soil. When knockdown (KD)-35

PiSulT-P.indica colonized with the plant, it results in an 8-fold reduction in the transfer of sulphate 36

to the colonized plants as compared to the plants colonized with the WT S. indica, which suggests 37

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 8, 2020. ; https://doi.org/10.1101/2020.01.07.897710doi: bioRxiv preprint

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that PiSulT is playing a role in sulphate transfer from soil to host plant. Further, plants colonized 38

with the WT S. indica were found to be healthy in comparison to the plants colonized with the 39

KD-PiSulT-P.indica. Additionally, S. indica colonization provides a positive effect on total sulfur 40

content and on plant metabolites like sulfate ions and glutathione, particularly under low sulphate 41

condition. We observed that the expression of sulfur assimilation pathway genes of S. indica and 42

plant is dependent on the availability of sulphate and on the colonization with the plant. Our study 43

highlights the importance of PiSulT in the improvement of sulfur nutrition of host plant 44

particularly under low sulphate condition and in plant growth development. This study will open 45

new vistas to use S. indica as a bio-fertilizer in the sulphate deficient field to improve crop 46

production. 47

48

INTRODUCTION 49

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Sulfur is an essential macronutrient for plant growth development and plays a fundamental 51

role in metabolism. Sulfur is a structural component of protein disulfide bond formation, Fe-S 52

group of electron transport chain, amino acids (cysteine and methionine), vitamins (biotin and 53

thiamin), cofactors (S-adenosyl methionine) (Droux, 2004; Pilon-Smits and Pilon, 2007). Sulfur 54

deficiency leads to a decrease in protein biosynthesis, chlorophyll content and eventually loss of 55

crop yield (Sexton et al., 1997; Buchner et al., 2004; Lunde et al., 2008; Davidian and Kopriva, 56

2010). Sulfur contributes around 0.1% of the earth's crust but it is very less accessible to living 57

beings (Kertesz, 2000). Plants utilize sulfur primarily in its anionic form (SO42−), which is 58

generally available in very less amount in the soil. As sulphate is water-soluble, therefore it 59

quickly loses from the soil by leaching (Eriksen and Askegaard, 2000; Buchner et al., 2004; 60

Davidian and Kopriva, 2010). Under the condition of low sulfur availability in soil, a symbiotic 61

association of an arbuscular mycorrhizal fungus (AMF) can help host plants to fetch sulfur from 62

the soil. It has been established that a fungal partner helps host plant roots in nutrients uptake from 63

nutrient-depleted soil rhizosphere, and in response, the fungal partner gets a carbon source from 64

plants (Parniske, 2008). In this association, fungal nutrient transporter helps in nutrient transfer to 65

host plant. It has been reported that the colonization of AMF Glomus intraradices can reduce 66

sulfur starvation in plants like Medicago truncatula (Sieh et al., 2013). It has also been reported 67

that AM symbiosis with the plant not only helps in nutrients uptake but also in the detoxification 68


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of metal contamination. For instance, Rhizophagus irregularis helps M. truncatula in the sulfur 69

acquisition as well as in the chromium detoxification (Wu et al., 2018). It has been observed that 70

sulphate transporter induced by both sulfur starvation and mycorrhiza formation show improved 71

sulphate concentration in Lotus japonicus (Giovannetti et al., 2014). Arbuscular mycorrhizal 72

colonization of G. etunicatum, G. intraradices with Allium fistulosum plants appears to make a 73

substantial contribution to the sulfur status (Guo et al., 2007). It has been reported that AMF like 74

G. intraradices helps in sulphate uptake and its translocation in the case of carrot especially under 75

low sulphate condition (Allen and Shachar-Hill, 2009). Till date studies on sulphate transport in 76

fungi have been limited to a few species such as Saccharomyces cerevisiae, Neurospora crassa, 77

Penicillium chrysogenum, and Aspergillus nidulans (Breton and Surdin-Kerjan, 1977; Cherest et 78

al., 1997; Marzluf, 1997; Van De Kamp et al., 1999; Van De Kamp et al., 2000; Piłsyk et al., 79

2007). However, due to the absence of a suitable transformation system in case of AMF, their 80

sulphate transporter gene could not be genetically manipulated to improve sulfur uptake in plants 81

colonized with AMF. Hence, mycorrhizal sulfur transfer to the host plant was poorly understood. 82

S. indica was isolated from the rhizosphere soils of the woody shrubs Prosopis juliflora 83

and Zizyphus nummularia from the sandy desert soils of Rajasthan, northwest India (Verma et al., 84

1998). It has a typical pear-shaped chlamydospore and belongs to the newly formed order 85

Sebacinales of Basidiomycota (Weiß et al., 2016). The size of S. indica genome is 24.97Mb 86

having 1884 scaffolds and 2359 contigs (Zuccaro et al., 2011). S. indica has a wide range of host 87

plants from bryophytes to angiosperms and monocots to dicots (Qiang et al., 2012). It colonizes 88

the root of several economically important plants like rice, barley, wheat and showed mutualistic 89

association with host plants (Jogawat et al., 2016). Unlike AMF, S. indica can be cultivated 90

axenically and based on a well-established transformation system, studies have been conducted to 91

understand the function of various genes in S. indica (Yadav, 2010; Akum et al., 2015). 92

Association of S. indica with host plants provides several beneficial impacts to the host plant such 93

as growth development, and it's coping up with biotic and abiotic stresses (Waller et al., 2005; 94

Kumar et al., 2009; Yadav, 2010; Johri et al., 2015; Jogawat et al., 2016; Narayan et al., 2017). 95

Because of these qualities, S. indica has termed as plant probiotic (Aschheim et al., 2005). It has 96

been reported that S. indica helps the colonized plants by the acquisition of nutrients such as 97

phosphorus, magnesium, and iron from nutrient-deprived soil rhizosphere with the help of its 98


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nutrient transporters viz., phosphate, magnesium and iron transporter respectively (Yadav, 2010; 99

Prasad et al., 2018, Verma et al 2019). 100

Characterization of sulphate transporter would provide insight into the regulation of 101

sulphate uptake during symbiosis. In the present study, PiSulT has been, identified, isolated, 102

functionally characterized and its role has been investigated in the sulphate transfer to the host 103

plant. We demonstrate that PiSulT is essential for sulphate transfer to the host plant and helps in 104

plant growth and development particularly under low sulphate condition. Additionally, this is the 105

first report of a complete analysis of the regulation of sulphate assimilation pathway genes of S. 106

indica and maize plants during colonization. Our results show that the biosynthetic steps are 107

regulated at the levels of mRNA expression during adaptation under low sulphate condition. We 108

suggest that the use of S. indica not only complements crop growth strategies but may also serve 109

as a model system to study molecular mechanisms related to indirect uptake of sulphate by the 110

plants and its regulation. 111

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RESULTS 113

114

Identification and cloning of PiSulT: 115

Our In-Silico analysis showed that putative sulphate transporter of S. indica belongs to 116

PIRI_contig_0011 (Accession no CCA67103.1) in S. indica genome (Zuccaro et al., 2011). It has 117

been annotated as probable sulphate permease, S. indica DSM11827. S. indica PiSulT shares 42% 118

and 49% sequence identity and highest query coverage of 90% and 73% with S. cerevisiae 119

sulphate transporters, Sul1, and Sul2 respectively. To amplify the putative PiSulT gene, total RNA 120

isolated from the S. indica and further cDNA library was constructed. This cDNA was used as a 121

template to amplify PiSulT with the help of gene-specific primers. The PCR amplified fragment 122

was cloned into a pJET1.2 vector and further confirmed by sequencing and this fragment was sub-123

cloned into pYES2 yeast shuttle vector. We found PiSulT is 2292 bp long ORF. A deduced amino 124

acid sequence of a putative PiSulT protein contains 763 amino acids and predicted polypeptide has 125

a molecular weight of approx. 83.2 kDa. Sequences comparison showed that PiSulT has 7 exons 126

and 6 introns (Supplemental Figure 1, 2 & 3). 127

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Phylogenetic and homology analysis: 130

131

Our conserved domain analysis showed the presence of all important domains such as 132

STAS (533-660), Sulphate_transp (164-455), Sulphate_tra_GLY (50-132), SUL1 (44-650) and 133

PRK11660 (38-429) in PiSulT (Supplemental Table 1). It was also found that putative PiSulT 134

has all the important motifs, domains, and sites that are essential for a protein to be defined as an 135

MFS transporter. Importantly, we observed that relative spatial positions of STAS and catalytic 136

domain in the case of S. indica PiSulT and S. cerevisiae Sul1 & Sul2 are the same. MultiAlin 137

alignment of the deduced amino acid sequence of PiSulT with the amino acid sequence of sulphate 138

transporter of a different kingdom and different group of fungus showed high and low consensus 139

peptide sequence with highly conserved amino acids sequences at each position. The signature 140

motif “GLY” of sulphate transporters is shown in the box and other conserved domains are shown 141

in red shaded regions (Supplemental Figure 4 and 5). 142

Phylogenetic analysis with diverse groups such as bacteria, insects, mammals, plants and 143

fungi members were constructed to understand the position of putative PiSulT among fungi 144

members and other groups. It was found that PiSulT is close to a member of Basidiomycota 145

(Figure 1). PiSulT shares 42%, 33%, 30% and 29% sequence identity with sulphate transporter of 146

Saccharomyces cerevisiae, Homo sapiens, Drosophila melanogaster and Arabidopsis thaliana 147

respectively. Low sequence identity (27%) was observed with prokaryotic sulphate transporters 148

such as E. coli and highest with fungal transporter (Table 1). Putative PiSulT showed the highest 149

similarity to sulphate permease of fungus Serendipita vermifera (75%) and Rhizoctonia solani 150

(65%) (Supplemental Table 2). It has more similarity with sulphate permease homolog protein of 151

the member of Basidiomycota than the Ascomycota. (Supplemental Table 3). 152

153

PiSulT expresses more under low sulphate condition: 154

155

To study the effect of sulphate concentration on PiSulT expression, S. indica culture were 156

grown in MN medium containing different concentrations (1µM, 5µM, 10µM, 25µM, 50µM, 157

100µM, 1mM, and 10mM) of sulphate (Na2SO4). The S. indica was harvested at 1, 5, 10, and 15 158

days and RNA were isolated. The expression pattern of the PiSulT gene was determined by 159

quantitative real-time PCR and semi-quantitative PCR. An increased expression level of PiSulT 160


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was observed at all the time points when sulphate was supplied at a concentration below100 µM 161

(Figure 2A & 2B). This increased expression of PiSulT under low sulphate condition indicates the 162

high-affinity nature of PiSulT. 163

164

Interaction of S. indica with maize plant: 165

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We found a maximum of 70 % colonization of the S. indica in the roots of the maize plant 167

at the end of 20 days. Colonization was confirmed by histochemical analysis (Figure 2C). We 168

found that as the colonization of S. indica increases in the root, a gradual increase in the expression 169

of PiSulT also takes place (Figure 2C and 2D). Additionally, it was observed that colonization is 170

associated with the developmental stage of the host tissue. S. indica showed strong colonization 171

with newly formed lateral roots than tap roots. Heavy intercellular colonization in cortical tissue of 172

differentiation and elongation zone was observed. No colonization was observed in the root tip 173

meristem including root cap (Supplemental Figure 6). To validate this finding, we determined the 174

amount of S. indica in different root zones by semi-quantitative PCR using S. indica genomic 175

DNA as a template for the quantification of the S. indica translation elongation factor gene (Tef). 176

A strong band intensity of Tef in the maturation zone was observed. However, a low-intensity 177

band was observed in the apical zone (Supplemental Figure 6i). 178

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Complementation assay and growth analysis: 180

181

Heterologous functional expression of PiSulT was analyzed in a yeast mutant cell of 182

sulphate transporter HK14 (∆sul1∆sul2) by complementation and growth assay. For the 183

complementation assay, PiSulT was cloned into a pYES2 yeast expression vector and transformed 184

into HK14 mutant cells. For positive control, BY4742, a parental/WT strain of HK14 was used. 185

This BY4742 cell contains both high-affinity sulphate transporter sul1 and sul2. For negative 186

control, mutant HK14 cells were transformed with empty vector pYES2. It is important to note 187

that pYES2 has a galactose-inducible promoter, therefore, it can only express in the presence of 188

galactose. Complemented mutant cells were tested to grow on the glucose and galactose 189

supplemented media to confirm the controlled and regulated expression of a pYES2 vector having 190

the PiSulT gene. It was observed that WT (BY4742) grew well on both glucose as well as 191


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galactose supplemented with the sulphate as they have a WT sulphate transporter gene. However, 192

no growth was observed in the case of mutant transformed with the empty vector under both the 193

conditions due to the lack of sulphate transporter. Mutant HK14 transformed with the PiSulT 194

found to grow in the galactose only because of the induction of pYES2 vector by galactose and 195

product of this gene restore the sulphate transport in HK14 cells (Figure 3Ai and 3Aii). 196

Therefore, we conclude that PiSulT functionally complements the HK14 mutant. The growth 197

pattern of all the above three types of cells at different concentrations of sulphate from low to high 198

was also analyzed (Figure 3Bi and Bii). A similar growth pattern was observed in the case of WT 199

and complemented strain, at all the concentrations of sulphate. However, in case of control (a 200

mutant transformed with the empty vector), no growth was observed (Figure 3Biii). Nevertheless, 201

the complemented strain showed the same growth pattern as of WT which indicates that the 202

transformed PiSulT restore the sulphate transport activity in the mutant cells similar to that of WT. 203

204

Chromate toxicity test to confirm the sulphate transport role of PiSulT: 205

206

It has been established that chromate enters into the yeast cells through sulphate 207

transporter. Transport of sulphate and chromate is a type of competitive transport, and it depends 208

on the concentration of either of the substrates. To confirm the role of PiSulT in sulphate transport, 209

we have performed the drop test. For this purpose, chromate toxicity was analyzed in the presence 210

of different concentrations of sulphate. We observed that when the concentration of sulphate 211

increases from 100µM to 1mM (with a constant concentration of chromate 20µM), there is a relief 212

from chromate toxicity at higher concentrations of sulphate (Supplemental Figure 7). Further, 213

WT, mutant and complemented HK14 were spotted on YNB plates containing an increasing 214

concentration of chromate i.e. 40µm and 60µM. In the case of control, no chromate was used. 215

Mutant strain, which does not have any sulphate transporter gene showed resistant to chromate and 216

grew well. However, WT and complemented strain having sulphate transporter genes were 217

susceptible to chromate toxicity and as a result, very little growth was observed (Supplemental 218

Figure 8). This observation supports the sulphate transport nature of PiSulT gene. 219

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Sulphate uptake and kinetic analysis: 223

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For the functional characterization of PiSulT, the following sets were used (a) WT 225

(BY4742) (b) mutant transformed with empty vector pYES2 (used as a control). (c) mutant 226

transformed with PiSulT (Figure 4A). It was observed that 296 pmol of sulphate was transferred 227

in the case of WT and 146 pmol in the case of complemented mutant. However, a negligible 228

amount i.e., 0.2 pmol was found to be transferred by the mutant transformed with the empty vector 229

(Figure 4A). The sulphate uptake by complemented HK14 cells expressing PiSulT follows typical 230

Michaelis-Menten kinetics with an apparent Km of 15.0675±1.75 µM and Vmax value of 231

1.917±.063 pmol/min/A650 (Figure 4B). The Km of 8.2±1.38 µM and Vmax of 3.204±.041 232

pmol/min/A650 was observed in WT cells. To obtain the optimum pH value for the function of 233

PiSulT, the mutant transformed with PiSulT was subjected to 35S-sulphate transport at different pH 234

values ranging from pH 2 to 8. We found that sulphate transport activity of PiSulT is pH-235

dependent. The optimum value for sulphate transport by PiSulT was found to be pH 5 (Figure 236

4C). 237

238

Role of PiSulT in sulphate transfer to host plant: 239

240

To know the role of PiSulT in sulphate transfer to the host plant, knock-down sulphate 241

transporter strain of S. indica (KD-PiSulT-P.indica) was developed by using RNA interference 242

(RNAi). To knock-down PiSulT gene, we have used a special pRNAi vector having duel S. indica 243

promoter PiTEF and PiGPD (Supplemental Figure 9i). The knockdown strain was selected on 244

primary and secondary selection media containing Hygromycin as described in the method section 245

(Supplemental Figure 10A). The expression of PiSulT in knock-down strain was analyzed by 246

using qRT-PCR. It was found that PiSulT transcripts level was reduced in all obtained transformed 247

colonies. However, in the case of TC1 transcript level was found to be very less (Supplemental 248

Figure 10B). The values obtained for PiSulT expression for WT (control), TC1, TC2, TC3 and 249

TC4 were 1.0, 0.42, 0.68, 0.45 and 0.73-fold (~60 % decrease in case of TC1) respectively relative 250

to PiTef. Furthermore, TC1 showed the highest silencing of PiSulT expression as compared to 251

other transformants and WT S. indica, hence selected for further experiments (Supplemental 252

Figure 10B). Further, the presence of the RNAi construct in knockdown S. indica was confirmed 253


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by PCR using hygromycin gene-specific primers. Amplification of a band was observed in all four 254

transformants except WT S. indica (Supplemental Figure 10C). The siRNA accumulation was 255

analyzed in the case of WT and KD-PiSulT-S. indica. We have observed the accumulation of 256

siRNA in the case of KD-PiSulT S. indica. However, no detection of siRNA was observed in the 257

case of WT (Supplemental Figure 10D). The growth of TC1 was also analyzed in KF broth and 258

on KF agar plates. Both WT and TC1 colony grow in a similar fashion on KF media without 259

Hygromycin. However, no growth of WT S. indica was observed as compared to TC1 when grown 260

in KF supplemented with Hygromycin (Supplemental Figure 11A and 11B). 261

The participation of S. indica in the transportation of sulphate from surroundings media to 262

host plant was confirmed by using bi-compartment assay (Supplemental Figure 12). In the first 263

set (set a) autoradiography revealed extensive labeling of maize plants by uptake of radiolabelled 264

35S in the case of WT S. indica (Figure 5Ai and 5Aii). The 35S was transferred to maize plants 265

through the fungal mycelium and across the hyphal bridge between both compartments. Very little 266

radioactivity was observed in the agar media of the second compartment confirming that the 267

amount of 35S present in the maize plants was exclusively transferred by S. indica and not because 268

of leaching by the fungus in the second compartment. In the case of set b, very less radioactivity 269

was detected in maize plants colonized with KD-PiSulT-P.indica transformant, confirming direct 270

role by PiSulT in sulphate transport to maize plants (Figure 5Bi and 5Bii). In the case of set c, no 271

radioactivity was observed (Figure 5Ci and 5Cii) hence, the movement of 35S from one chamber 272

to another was not due to diffusion but by the fungus only. We have observed that 362 pmol of 273

sulphate was transported by WT S. indica to the host plant as compared to the 43 pmol in the case 274

of KD-PiSulT-S. indica and this difference was found to be statistically significant (p<0.01) 275

(Figure 5D). Colonization of both WT and KD-PiSulT- S. indica into maize plants was found to 276

be similar in both the cases, i.e. 70 % at 20 dpi (Figure 5Aiii and 5Biii). It is important to note 277

that in the colonized state, S. indica has external and internal hyphae. External hyphae ramify out 278

of the colonized root and internal hyphae penetrate the root cortex. To determine the expression of 279

PiSulT in internal and external hyphae, transcript abundance was measured by using relative 280

quantitative RT-PCR. It was observed that PiSulT transcripts were 2.25 folds higher in the external 281

hyphae as compared to internal hyphae and this was found to be statistically significant (P<0.05) 282

(Figure 5E). 283

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Plant responses to sulfur deficiency and the role of PiSulT in sulphate nutritional 285

improvements of the host plant: 286

287

The role of PiSulT in sulphate nutritional improvement of the host plant was analyzed. 288

Plants colonized with WT S. indica were found to be healthy as compared to the non-colonized 289

plants and plants colonized with the KD-PiSulT-S. indica, grown under low sulphate condition 290

(Figure 6A). Under similar conditions, biomass (fresh weight) of maize plants colonized with WT 291

S. indica was found to be 1.8 fold and 2.4 fold higher and sulphate contents were found to be 1.6 292

and 2.3 fold in comparison of plants colonized with the KD-PiSulT-P.indica and non-colonized 293

plants respectively and this was found to be statistically significant (p<0.001) (Figure 6B and 294

6C). In a separate study to know the performance of the S. indica in the growth promotion activity 295

of the plants, the plant responses under low sulphate condition (10µM) and sulphate-rich (10mM) 296

conditions were chacked. For this purpose, four sets were prepared: (1) maize plants grown under 297

low sulphate condition and treated with autoclaved macerated fungal mycelium (served as a 298

control) (2) maize plants colonized with WT S. indica and grown at low sulphate condition, (3) 299

maize plants grown under high sulphate condition and treated with macerated fungal mycelium 300

(served as a control) (4) maize plants colonized with WT S. indica and grown at high sulphate 301

condition. All four experimental sets were grown in acid-washed sand fertilized with a modified 302

0.5X Hoagland solution (Hoagland and Arnon, 1950) containing respective sulphate 303

concentrations. After 4 weeks, plants were harvested, and fresh weight was measured. We 304

observed that biomass (in terms of fresh weight) of maize plants colonized with WT S. indica was 305

1.2 fold higher when grown at sulphate-rich condition, whereas it was 2.3 fold higher in the case 306

of maize plants colonized with WT S. indica and grown under low sulphate condition in 307

comparison to their respective controls (p< 0.05) (Figure 6D). The total sulfur content was 308

estimated in such conditions and, it was observed 2.3 and 1.6 fold high under similar conditions 309

(Figure 6E). We also analyzed the content of plant metabolites such as glutathione and sulphate 310

ions in plants under similar conditions during colonization. Glutathione content was found to be 311

1.8 and 0.8 fold and sulphate ions were found to be 1.5 and 0.6 fold higher under similar 312

conditions (Figure 7 A & B). 313

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Expression analysis of sulphate assimilation pathway genes of S. indica grown axenically and 317

during colonization with the host plant: 318

319

The up and down-regulation of sulfur assimilation genes were observed in case of WT and 320

KD-PiSulT-S. indica that was grown axenically under low sulphate condition (Supplemental 321

Figure 13; Supplemental Table 4). In case of WT S. indica, out of 22 selected genes from 322

sulphate assimilation pathway, 11 genes; siroheme synthase (PiMET1), sulfite reductase cys-4 323

(PiMET5), sirohydrochlorin ferrochelatase (PiMET8), methylenetetrahydrofolate reductase 324

(PiMET12), adenylylsulphate kinase (PiMET14), 3`-phospho- adenylylsulphate reductase 325

(PiMET16), O-acetyl homoserine (thiol)-lyase (PiMET17), bisphosphate-3'-nucleotidase 326

(PiMET22), sulphate adenylyltransferase (PiATPS), cystathione gamma-lyase (PiCYS3) and 327

sulphate transporter (PiSulT) were found to be up-regulated. Amongst two genes i.e., PiSulT and 328

PiMT16 were found to be 14 and 9.7 fold upregulated. In the case of KD-PiSulT-P.indica, 6 329

genes were found to be up-regulated i.e., transcription factor (PiMET4), 22-fold, 330

methylinetetrahydrofolate reductase (PiMET12), 5.1-fold, methylenetetrahydrofolate reductase 331

(PiMET13), 10-fold, adenylyl-sulphate kinase (PiMET14) 10.7-fold and sulphate transporter 332

(PiSulT) 4.1-fold. A very high i.e., 48.6-fold up-regulation was found in the case of cyctathionine 333

gamma-lyase (PiCYS3) (Supplemental Figure 14; Supplemental Table 4). Only 3 genes i.e., 334

siroheme synthase (PiMET1), 3`-phospho- adenylylsulphate reductase (PiMET16), and PiSulT 335

were found to be up-regulated during colonization of the WT S. indica with the maize plant grown 336

under low sulphate condition. A maximum of 12-fold up-regulation was found in the case of 337

PiSulT under similar conditions (Supplemental Figure 15; Supplemental Table 4). When KD-338

PiSulT-S. indica was colonized with the maize plants, 6 genes, i.e., sulfite reductase flavin-binding 339

alpha-subunit (PiMET10), methylinetetrahydrofolate reductase (PiMET12), 340

methylenetetrahydrofolate reductase (PiMET13) and sulphate adenylyltransferase (PiATPS), were 341

found to be up-regulated and a maximum 24-fold, up-regulation was found in case of PiMET1. 342

Interestingly, PiSulT was found to be down-regulated under similar condition as compared to WT 343

S. indica (Supplemental Figure 16; Supplemental Table 4). 344

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Expression analysis of sulphate assimilation pathway genes of maize plants during 350

colonization with WT and KD-PiSulT-S. indica: 351

352

In case of maize plants colonized with WT S. indica and grown under low sulphate 353

condition, out of 31 selected genes of sulphate assimilation pathway, only 3 genes i.e., 354

Methylthioadenosine nuclease (ZmMTN), serineacetyltransferase2 (ZmSAT2) and 355

sulfotransferase (ZmSOT) were found to be up-regulated by 2-5-fold (Supplemental Figure 17; 356

Supplemental Table 5). In the case of plants colonized with the KD-PiSulT-S. indica, 13 genes 357

were found to be up-regulated. A maximum of 6.6 and 6.2-fold up-regulation was found in the 358

case of serine acetyltransferase1 (ZmSAT1) and gamma-glutamyltransferase1 (ZmGGT1), 359

respectively (Supplemental Figure 18; Supplemental Table 5). We have also found the up-360

regulation of APS kinase (ZmAPSK). Interestingly, all three sulphate transporter genes; sulphate 361

transporter1 (ZmST1), sulphate transporter (ZmST3.4) and sulphate transporter (ZmST4.1) were 362

found to be down-regulated in plants either colonized with the WT or KD-PiSulT-P.indica 363

(Supplemental Figure 17 and 18; Supplemental Table 5). 364

365

DISCUSSION 366

367

The rhizosphere is observed as a hot spot for microbial activity. Microbes like bacteria, 368

saprophytes, and mycorrhizal fungi, helps in nutrient enrichment for plants by mobilization and 369

cycling of nutrients. Due to leaching, high reactive nature, complex forms and insolubility, many 370

nutrients are unavailable to the plants. Hence, plants have developed many strategies to cope with 371

nutrients deficiency including sulphur. For example, modulation of the root system architecture 372

such as root length, modulation of transport activity with distinct transport affinities, substrate 373

specificities to ensure appropriate flux (Aibara and Miwa, 2014). Low availability of nutrients like 374

sulphate, phosphate and iron results in the less crop yields all over the world. Amongst nutrients 375

sulphate is also play important role in the plant growth and development and its deficiency also 376

causes less crop production. It has been reported that soil texture and rain are the major factors that 377

affect sulfur availability in the soil. Sandy and silty soil have less organic matter and often low in 378

sulfur because a high rainfall leaches out sulphate very easily from the root zone (Scherer, 2009). 379

Sulphate is the main sulfur source for plants contributing to about 5% of total soil sulfur. 380

Generally, more than 95% of soil sulfur are organically bounded (sulphate ester and 381


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carbon‐bonded) and thus are not directly available for plants (Fitzgerald, 1976; Tabatabai, 1986; 382

Leustek, 1996; Scherer, 2001; Scherer, 2009; Gahan and Schmalenberger, 2014). It has been 383

observed that plants require sulfur concentration between 0.1 to 0.5% of dry weight and 384

concentration below 0.1% is critical for the normal plant growth (Daigger and Fox, 1971; Kang 385

and Osiname, 1976; Kamprath and Jones, 1986; Sakal et al., 2000; Marschner, 2011; Sutar et al., 386

2017). The optimum range of soil sulphate content considers from 0.3% to 1.0% of the dry weight 387

of the soil (Little and Nair, 2009). A concentration of 3-5 ppm of sulphate in the soil is adequate 388

for the growth of many plant species including maize (Sutar et al., 2017). Most of the sulphur in 389

soil is present in an organically bounded form, which is released by the enzymatic action of 390

bacteria and fungi, therefore, becomes available for utilization by plants (Kertesz and Mirleau, 391

2004; Gahan and Schmalenberger, 2014; Speck, 2015; Jacoby et al., 2017). Due to the low 392

availability, sulfur is the limiting factor for crop production throughout the world and is a major 393

challenge for agriculture. It has been reported that induction of the sulphate sensing and expression 394

of high-affinity sulphate transporter is the key mechanism to increase the sulphate uptake rate in 395

roots under low sulphate conditions (Takahashi, 2019). All the above strategy works only when 396

plant roots are in contact with the nutrients in the rhizosphere. But in case of nutrient-depleted soil, 397

another important strategy of plant-fungal association can work to take nutrients from nutrients 398

deprive soil with the help of fungal nutrient transporter. In this study, functional characterization 399

of a high-affinity sulphate transporter from S. indica and its role in the transfer of sulphate to the 400

host plant has been demonstrated and how PiSulT is helpful to the plant growth and development 401

under low sulphate condition has been established. 402

We found that PiSulT showed the highest similarity with the sulphate permease of S. 403

vermifera and Rhizoctonia solani among the fungus group. Phylogenetic analysis indicates that 404

PiSulT is more closely related to high-affinity sulphate transporter of fungi. Functional 405

domains/motifs analysis of PiSulT polypeptide indicates that it is a sulphate transporter membrane 406

protein having a typical C- terminal STAS domain, a signature motif of sulphate transporter 407

“GLY” and a catalytic domain which is essential for a protein to be a sulphate transporter. It has 408

been reported that in eukaryotes STAS domain plays an important role in sulphate transport and 409

post-transcriptional regulation (Rouached et al., 2005; Yoshimoto et al., 2007). Additionally, 410

sulphate permease signature motif was also identified in case of PiSulT polypeptide, the similar 411

motif was also reported in case of sulphate transporter of fungus and plant thus support our data 412


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(Sandal and Marcker, 1994; Smith et al., 1995; Van De Kamp et al., 1999). We have observed 413

similar percent colonization of S. indica in case of maize plant either grown under low or high 414

sulphate condition which suggests that colonization is not dependent on the sulfur availability. 415

Further, it was found that the colonization pattern of S. indica with a host plant was associated 416

with the developmental stage of root tissue. We found that maturation and differentiation zone of 417

lateral roots was densely colonized in comparison to the distal part of the apical root meristem. A 418

similar pattern of colonization was also observed in the case of S. indica colonized with barley 419

plants (Deshmukh et al., 2006), hence support our data. 420

A high transcript level of PiSulT under low sulfate concentrations (< 100 µm) indicates the 421

high-affinity nature. Similar observations were also made in case of high-affinity phosphate 422

transporter of algae, fungi and sulphate transporter of S. cerevisiae, therefore, these studies support 423

our data (Chung et al., 2003; Yadav, 2010; Kankipati et al., 2015). 424

As our growth analysis showed that PiSulT complemented mutant and WT have similar 425

and typical diauxic growth pattern which suggests that PiSulT efficiently complements the mutant. 426

Previously, it has been shown that chromate enters the cells mainly through sulphate transporter 427

and competitively inhibits sulphate uptake (Pereira et al., 2008). Several studies have been 428

reported for uptake of chromate by sulphate transporter in different organism like in yeast, fungi, 429

bacteria, and in mammalian cells (Ohta et al., 1971; Roberts and Marzluf, 1971; Campbell et al., 430

1981; Smith et al., 1995a; Cherest et al., 1997). Transport of sulphate and chromate is a type of 431

competitive transport, and it depends on the concentration of either of the substrate. For this 432

purpose, chromate toxicity was analyzed in the presence of different concentrations of sulphate. 433

We observed that mutant (HK14) grew well and showed resistant to chromate as there was no 434

uptake of chromate due to the absence of sulphate transporter gene, however, a very less growth 435

was observed in case of WT and mutant complemented with the PiSulT (due to the uptake of 436

chromate), therefore both were found to be susceptible to chromate toxicity. This observation 437

confirms that PiSulT is a sulphate transporter. 438

Kinetics data reveals that PiSulT follows typical Michaelis-Menten kinetics. The apparent 439

Km value was found to be 15 µM. Kinetic analysis of the sulphate uptake isotherm obtained in a 440

range of low external sulphate concentrations (0–100µM) revealed that PiSulT has a high affinity 441

for sulphate similar to those of other high-affinity sulphate transporters having Km values ranges 442

from 4-14 µM (Smith et al., 1995b; Smith et al., 1995; Cherest et al., 1997; Smith et al., 1997; 443


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Takahashi et al., 2000; Vidmar et al., 2000; Yoshimoto et al., 2002; Howarth et al., 2003; Nocito 444

et al., 2006). To best of our knowledge high-affinity sulphate transporter has Km ~12 μM and low-445

affinity has a Km ~100μM (Piłsyk and Paszewski, 2009). Previously, two high-affinity sulphate 446

transporter of S. cerevisiae, have been reported Sul1 & Sul2 with a Km range between 4 to 10 447

µM (Smith et al., 1995a; Cherest et al., 1997). Therefore, we suggest that PiSulT belongs to high-448

affinity sulphate transporters. Our pH dependence analysis suggests that PiSulT functions 449

maximum at pH 5. The effect of pH on sulphate uptake suggests that PiSulT transport might be 450

facilitated by the proton gradient across the plasma membrane. 451

In order to know the role of PiSulT in sulphate transfer to the host plant, KD-PiSulT-452

P.indica were colonized with the host plant, this results in the reduction of the transfer of sulphate 453

to the colonized plants as compared to the plants colonized with the WT S. indica, which suggests 454

that PiSulT play a role in sulphate transfer from soil to host plant. A significantly higher 455

expression of PiSulT in external hyphae than internal hyphae was observed, which indicates that 456

external hyphae are the main site of PiSulT expression, and it is helping in the uptake of sulphate 457

(available outside) to plant roots. A similar finding was also observed in case of PiPT when maize 458

plants were colonized with the S. indica, this authenticates our data (Yadav, 2010). This 459

expression pattern of PiSulT suggests that S. indica is supportive in the acquisition of sulphate 460

from deprive range of sulphate concentration in soil rhizosphere with the help of hyphae. 461

Our study highlights the importance of PiSulT in the improvement of sulfur nutrition of the 462

host plant particularly below plant’s required sulphate concentration (0.1 to 0.5% of plant dry 463

weight) and below normal range of soil sulphate concentration (0.3% to 1.0% of dry weight of 464

soil) for adequate plant growth and development. We observed that in the case of WT S. indica 465

colonized plant biomass was 2.4 and 1.8-fold more than that of the non-colonized plants as well as 466

from the KD-PiSulT-P.indica colonized plants, respectively. Importantly, it was found that the 467

sulphate has an impact on the biomass of the maize plant colonized with S. indica. In our study, 468

total sulfur content and biomass were found higher in the plants colonized with WT S. indica as 469

compared with non-colonized and KD-PiSulT-P.indica-colonized plants, this suggests that 470

sulphate is playing a role in augmenting plant yield or biomass, and this increase in the biomass is 471

due to the PiSulT. Further, the effect of KD-PiSulT-S. indica colonization on plant metabolite 472

content shows that low sulphate availability results in the low level of metabolites and sulfur-473

containing compounds such as glutathione. In a study, it has been shown that sulphate starvation 474


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affects other metabolic pathways in a pleiotropic manner (Nikiforova et al., 2005; Sieh et al., 475

2013). Our study demonstrates improved sulphur nutrition and the importance of S. indica in 476

sulphur uptake for plant metabolism. It is interesting to note that the biomass promoting activity of 477

S. indica was more under low sulphate condition as compared to that of sulphate-rich condition. A 478

similar result was also found in the case of sulphate transporter of M. truncatula associated with 479

mycorrhizal fungi (Sieh et al., 2013), hence validate our data. 480

Sulphate transport is the initial step in the acquisition and assimilation of sulfur and the 481

flux of sulfur through the assimilatory pathway is likely to be linked to the regulation of sulphate 482

transporters. Transcriptional regulation of several genes encoding enzymes of the sulfur 483

assimilatory pathway in response to the plant sulfur status has also been reported (Droux, 2004; 484

Casieri et al., 2012). Our data suggest that the expression of sulfur assimilation genes is dependent 485

on the availability of sulphate. As we found more sulfur assimilation pathway genes were up-486

regulated during the KD-PiSulT-S. indica colonization with the plant, maybe these genes are 487

helping the plant in getting more sulphate from the soil. The assimilation of sulphate occurs 488

through a pathway that includes its uptake by specific permeases, activation of intermediates by 489

ATP-dependent adenylation and reduction to sulfite and further to sulfide. Sulphate assimilation 490

pathway genes particularly biosynthesis of methionine, cysteine, and S-adenosyl-methionine 491

(SAM) were found to be up-regulated in WT axenically grown S. indica which indicates the active 492

involvement of these genes in sulfur assimilation. The genes which are showing up-regulation in 493

the case of KD-PiSulT-P.indica (grown axenically) indicate that they are responsive to external 494

sulfur concentration and most probably involved in signaling under low sulphate condition. We 495

conclude that starvation of any one of several amino acids results in the increased expression of 496

genes encoding enzymes of multiple amino acid biosynthetic pathways. The down-regulation of 497

genes in the case of KD-PiSulT-S. indica can be explained by the effects of reduced sulfur 498

availability on the biosynthesis of amino acids, proteins, and sulfolipids. 499

In our study, many sulfur assimilation pathway genes appeared to be differentially 500

expressed upon sulfur deficiency between non-colonized and colonized plants. In the case of 501

maize plants colonized with WT S. indica and grown under low sulphate condition, out of 31 502

selected sulphate assimilation pathway-related genes, only 3 genes were found to be up-regulated 503

as compared to 13 genes of plants colonized with the KD-PiSulT-S. indica. It is established that 504

plants respond to a limited sulphate supply by increasing the expression of genes involved in the 505


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uptake and assimilation pathway. Amongst, 13 genes of a plant, we have found increased 506

expression of APR, APS kinase and SAT. The increased expression of APR and ATP-sulfurylase 507

indicates adapting response in the plant through the assimilatory pathway during the low supply of 508

the sulphate by the fungus to the plant. We hypothesize that when KD-PiSulT-P.indica provides 509

less sulphate to the plant during the colonization, in order to fulfill the high demand of sulphate in 510

the fast-growing roots, programmed responses got induced. Increased expression of APR, APS 511

kinase and SAT genes have been reported in case of maize plant under sulphate limited conditions 512

and authors have suggested that these enzymes are important regulatory components of the sulfur 513

assimilation pathway and got induced under sulphate-limited condition, therefore, helps the plant 514

during the sulphate deficiency, thus these reports support our data (Hopkins et al., 2004). Roots 515

growing in the soil may encounter glutathione originating from microbial activity or released by 516

organic matter. This exogenous glutathione pool may be a valuable source of reduced sulphur for 517

the fungi. It has been proposed that glutathione may play a role in the mycorrhizal symbiosis. 518

Recently, it was suggested that mycorrhiza can improve sulphate availability to the root cell by 519

extending their exploration horizon and, in exchange, receive reduced sulphur compounds from 520

the root cell (Mansouri-Bauly et al., 2006). GGT is known to promote the hydrolysis of 521

extracellular glutathione. As in our study, we found gamma-glutamyltransferase (ZmGGT1) 522

highly up-regulated during the interaction of KD-PiSulT-S. indica and maize plant which suggests 523

that glutathione will be available to the fungi in low quantity. We hypothesize that as the plant is 524

not getting an adequate supply of the sulphate, therefore not giving the reduced sulphur to the 525

fungi in exchange by increasing the expression of the ZmGGT1.Glutathione S-transferase (GST) 526

has been proposed to play an integral role in the plant defense against the toxins and found to be 527

induced during the chemical treatments and environmental stresses (Leustek et al., 2000). We 528

assume that in this case also when there is less supply of the sulphate to the host plant by the KD-529

PiSulT-S. indica, GST got induced to generate a plant defense mechanism to avoid any harsh 530

conditions. All other genes of sulfur assimilation pathway which were found up-regulated in case 531

of KD-PiSulT-P.indica colonization with the plant, which is working either as a precursor/ 532

regulatory component in the synthesis of amino acids or are involved as an intermediate in the 533

sulfur assimilation may be helping the plant to get more sulphate during deficiency therefore it 534

needs warrant investigation. 535

Our study also provides a new prospect to understand the sulphate transport network with 536


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18

and without a host plant. Additionally, the expression of the sulfur assimilatory pathway genes of 537

the maize plant and their significance in balancing sulfur flux to sulphate demand of the plant for 538

growth and development during interaction with the fungal partner will provide the insights into 539

the sulphate management by the plant during sulphate deficiency. Thus, we suggest that in future 540

S. indica can be used in the sulphate-deficient agriculture field to improve plant productivity. 541

542

METHODS 543

544

Plant, Fungi, Bacteria, and Yeast Strains: 545

546

Zea mays (HQPM-5) plant and fungus S. indica were used throughout the study. E. coli 547

XL1-Blue and DH5α were used for cloning purposes (Sambrook et al., 1989). Yeast sulphate 548

transporter (∆sul1&∆sul2) double mutant HK14 (MATα sul1::KanMX sul2::KanMX his3Δ leu2Δ 549

lys2Δ ura3Δ) and WT S. cerevisiae (BY4742) (MATα his3∆ leu2∆ lys2∆ ura3∆) were used for 550

complementation and kinetics (Kankipati et al., 2015) (Supplemental Table 6). Both strains have 551

the same BY background. Maize seeds were surface sterilized for 2 min in ethanol followed by 10 552

min in a NaClO solution (0.75% Cl) and finally washed six times with sterile water. Additionally, 553

seeds were also treated with double-distilled H2O at 60°C for 5 min and were germinated on water 554

agar plates (0.8% Bacto Agar, Difco, Detroit, MI) at 25 °C in the dark (Varma et al., 1999). Plants 555

were grown under controlled conditions in a greenhouse with an 8 hours light (1000 Lux)/ 16 556

hours dark period at a temperature of 28°C with a relative humidity 60–70%. Surface sterilized 557

pre-germinated maize seedlings were placed in pots filled with a mixture of sterile sand and soil in 558

the ratio of 3:1 (garden soil from Jawaharlal Nehru University campus and acid-washed riverbed 559

sand). Plants were weekly supplied with half-strength modified Hoagland solution containing the 560

following: 5 mM KNO3, 5 mM Ca(NO3)2, 2 mM MgSO4, 10 μM KH2PO4, 10 μM MgCl2, 4 μM 561

ZnSO4, 1 μM CaSO4, 1 μM NaMoO4, 50 μM H3BO3. Plant roots were harvested at different time 562

points after inoculation and were assessed for colonization. To study colonization, ten root 563

samples were selected randomly. Samples were softened in 10% KOH solution for 15 min and 564

acidified with 1 N HCl for 10 min and finally stained with 0.02% Trypan blue (Phillips and 565

Hayman, 1970; Dickson, 1998; Kumar et al., 2009). After 2 hours, samples were de-stained with 566

50% Lactophenol for 1–2 hours before observation under a light microscope (Leica Microscope, 567

Type 020-518.500, Germany and Nikon Eclipse Ti). The distribution of chlamydospores within 568


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the root was taken as an index for studying colonization. Percent colonization was calculated for 569

the inoculated plants according to the method described previously (McGonigle et al., 1990; 570

Kumar et al., 2009). 571

572

Isolation of PiSulT cDNA: 573

574

Total RNA was isolated from S. indica grown axenically in KF medium (Hill, 2001) 575

containing low sulphate (10µM) concentration with Trizol reagent (Invitrogen, USA) and cDNA 576

was synthesized using a cDNA synthesis kit (Stratagene). PiSulT ORF (2292bp) was PCR 577

amplified by using gene-specific primers (Supplemental Table 7). For directional cloning BamHI 578

and XbaI sites were added in gene-specific forward and reverse primers respectively 579

(Supplemental Table 7). For PCR reaction, S. indica cDNA was used as a template. PCR 580

reactions were carried out in a final volume of 50 μl, containing 10 mM Tris-HCl (pH 8.3); 50 mM 581

KCl; 1.5 mM MgCl2; 200 μM of dNTPs; 3 μM of each primer; 3 units of Phusion High-582

Fidelity DNA polymerase (Thermo Fisher Scientific and 60-100 ng of cDNA as template. PCR 583

program was used as follows: 94°C for 2 min (1 cycle), 94°C for 45 sec, 60°C for 1 min 15 sec, 584

72°C for 1-2 min (35 cycles) and 72°C for 5 min (1 cycle). The PCR product was cloned into a 585

pJET1.2 cloning vector (Promega) and further subcloned into the pYES2 yeast shuttle vector 586

between BamH1 and Xba1 sites. 587

588

Quantitative RT-PCR analyses: 589

590

S. indica culture was grown in MN medium. Following contents were used / liter (MgCl2, 591

731 mg; Ca(NO3)2.4H2O, 288mg; NaNO3, 80 mg; KCI, 65 mg; Glucose, 10 g; NaFeEDTA, 8 mg; 592

KI, 0.75 mg; MnCI2.4H2O, 6 mg; Zn Acetate, 2.65 mg; H3BO3, 1.5 mg; CuCl2,0.13 mg; 593

Na2MoO4.2H2O, 0.0024 mg; Glycine, 3 mg; Thiamine Hydrochloride, 0.1 mg; Pyridoxine 594

Hydrochloride, 0.1 mg; Nicotinic Acid, 0.5 mg; Myoinositol, 50 mg; Na2SO4 as per need, pH 5.5) 595

(Bécard and Fortin, 1988). Further, S. indica culture were harvested at different time points and 596

total RNA was isolated. The first strand of cDNA was synthesized with the Superscript cDNA 597

synthesis kit (Clontech) from 3g of total RNA and used as a template for PCR with gene-specific 598

primers (Supplemental Table 7). The reaction mixture was heated at 95 °C for 20 min and then 599

subjected to 40 PCR cycles of 95 °C for 3s, 65 °C for the 30s, and 72 °C for 20 s. The heat 600

dissociation curves confirmed that a single PCR product was amplified. The melting temperatures 601


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20

were 60-65 °C for the PCR products of the PiSulT. S. indica translational elongation factor gene 602

(PiTef) was used as a control (Yadav, 2010). The level of target mRNA, relative to the mean of the 603

reference housekeeping gene, was calculated by the relative ΔΔCt method as described by the 604

manufacturer. 605

606

Phylogenetic and homology analysis: 607

608

The functional sites in PiSulT and their pattern were determined using the PROSITE 609

database. For identification purposes, blastX algorithm (www.ncbi.nlm.nih.gov) was used. 610

Sequence alignments were done with ClustalΩ and BLOSUM62 with a gap penalty of 10 for 611

insertion and 5 for extension (Henikoff and Henikoff, 1992; Thompson et al., 1994). Phylogenetic 612

and molecular evolutionary analyses of S. indica putative PiSulT were constructed using MEGA X 613

with the neighbor-joining analysis examined by bootstrap testing with 1000 repeats (Kumar et al., 614

2018). 615

616

Complementation assay and growth analysis: 617

618

For this purpose, S. cerevisiae WT BY4742 and yeast high-affinity sulphate transporter mutant 619

strain HK14 were used (Kankipati et al., 2015). HK14 was transformed with a recombinant 620

pYES2 vector having PiSulT by LiCl-PEG method (Bun-Ya et al., 1991; Riesmeier et al., 1992; 621

Gietz et al., 1995; Akum et al., 2015; Jogawat et al., 2016). Yeast cells were grown at 30ºC on SD 622

media containing 0.1 mM of sulphate as a sole source of sulfur in the presence of 2% glucose 623

(non-inducing condition) and 2% galactose (inducing condition) as sole carbon source separately. 624

Cells were suspended in sterile distilled water and cell density was adjusted to A600= 0.1, followed 625

by serial dilutions of 1/10. HK14 cells transformed with empty vector were used as a control. The 626

drop test was performed to check the complementation. 30 µl suspensions were plated on 627

solidified agar plates containing glucose and galactose separately. Plates were incubated for 2-3 628

days and the growth pattern of cells was analyzed. For the growth pattern of all three strains WT, 629

mutant HK14 complemented with PiSulT and mutant complemented with empty vector pYES2 630

were also analyzed separately in SD media supplemented with different sulphate concentrations. 631

For this purpose, cells were starved for sulfur source and then transferred to medium containing 632

different concentrations of sulphate as the sole source of sulfur. The flasks were kept at 30°C and 633

OD600 was measured to observe a comparison between the WT and the transformed mutant strains. 634


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21

Experiments were carried out in triplicates and were repeated thrice. 635

636

Sulphate uptake and kinetics assay: 637

Yeast cells were grown up to ODA600 of 1.5-2 in selective SD synthetic medium (YNB 638

media with 2% glucose, lacking uracil) at 300C for two days at 220 rpm in a metabolic shaker 639

(Infors, Switzerland). Exponentially grown cells were washed with autoclave ddH2O and then 640

transferred to sulfur starvation media, containing 2% of galactose (pH 5, with 50mM MES-KOH) 641

at 300C, 220 rpm for two days. Cells were harvested and resuspended at a cell density of 60 mg 642

(wet weight)/ml. To start sulphate uptake, 50 µl of cells (preincubated for 10 min at 300C) were 643

aliquoted and different concentration of sulphate (1µM, 2µM, 4 µM, 6 µM, 10 µM, 25 µM, 75 644

µM, and 100 µM) was used. For this purpose, 0.5mM [35S] sodium sulphate (specific activity of 645

2000 cpm/nmol or 0.9 Ci/mol of sodium sulphate) was used. After 4 min, uptake was stopped by 646

adding 5 ml of ice-cold sulfur starvation media. Cells were recovered on a glass microfiber filter 647

and washed three times with 5 ml of ice-cold sulphate starvation media by centrifugation at 5000g 648

for 5min at 40C. For the blanks, ice-cold sulphate starvation media was added before the addition 649

of [35S] sodium sulphate, and the cells were immediately filtered and washed. Further, filters were 650

transferred into scintillation vials containing 5 ml of scintillation cocktail ‘O’ (CDH) and the 651

radioactivity was measured with a scintillation counter (Liquid Scintillation Analyzer TRI-CARB 652

2100TR; Packard). Uptake assay was performed at room temperature (250C). Sulphate 653

accumulation (in pmol) was measured by standard mathematical calculations to convert 654

scintillation count to pmole. The amount of sulphate transported by control (background) was used 655

to normalize the data. Transport data at 10µM concentration was used for plotting the uptake 656

graph. The rate sulphate uptake was expressed as nmol.min-1 x (mg dry weight)-1 or 657

pmol/min/A650. GraphPad Prism 6 was used to plot nonlinear regression for sulphate uptake rate. 658

Experiments were repeated three times and each time three replicates were taken. 659

660

Development of RNAi Cassette and knockdown S. indica: 661

662

A 452 bp unique fragment of PiSulT (Supplemental Figure 9 ii) was selected using the 663

BLAST tool and analyzed for its uniqueness and RNA 20 structures. This unique fragment was 664

amplified using the gene-specific primers (Supplemental Table 7) and cloned into a pGEM-T 665

cloning vector and subsequently subcloned into the pRNAi vector at the unique EcoRV site 666


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22

(Hilbert et al., 2012). This construct was named as pRNAi-PiSulT. Empty pRNAi and pRNAi-667

PiSulT was transformed into the S. indica as described previously (Yadav, 2010). In brief, 668

chlamydospores were harvested from 14 days old S. indica culture and were germinated under 669

glucose nutrition. For the cell wall disruption, β-Glucuronidase enzyme (Sigma: Helix pomatia) 670

was used. Linearized pRNAi-PiSulT (1µg) was transformed into S. indica using electroporation at 671

12.5 kV/cm, 25-microfarad capacitance and 5-ms pulse length. Four transformed colonies (TC1, 672

TC2, TC3, and TC4) were selected after primary and secondary selection using KF media 673

containing 100µM and 200 µM concentration of hygromycin respectively (Supplemental Fig. 674

10A). The transformation was confirmed by PCR using hygromycin gene-specific primers and 675

siRNA analysis (Supplemental Table 7; Supplemental Figure 10C & D). All four transformants 676

were tested for the expression of the silenced gene (PiSulT) by q-RT-PCR as described previously 677

(Jogawat et al., 2016). Transformants obtained were named as “KD-PiSulT-P.indica”. 678

679

Northern blot analysis: 680

681

Northern blot was performed for siRNA detection. For this purpose, total RNA was 682

isolated from KD-PiSulT-P. indica from the TC1 (in duplicate) and WT S. indica by using TRIzol 683

reagent and probe was prepared by end labeling of the PiSulT end labeling primer 684

(5′GTAATATCGACACGACCG) using [γ -32P] ATP and polynucleotide kinase as per the 685

instructions described in manual (Molecular Labeling and Detection, Fermentas). Hybridization 686

and autoradiography were performed as described (Yadav, 2010). RNA was dissolved in diethyl 687

pyrocarbonate (DEPC) water, heated to 65 °C for 5 min, and then kept on ice. To this, 688

polyethylene glycol (molecular weight of 8000, Sigma) was added to a final concentration of 5% 689

and NaCl to a final concentration of 0.5 M. After incubation on ice for 30 min, this mixture was 690

centrifuged at 10,000Xg for 10 min. The supernatant obtained was mixed with the three volumes 691

of ethanol. To precipitate the RNA, this mixture was kept at -20 °C for at 2 h. To obtain low 692

molecular weight RNAs, the mixture was centrifuged for 10 min at 10,000Xg. The pellet obtained 693

was dissolved in DEPC treated water and heated at 65 °C for 5 min. Further, one-third volume of 694

4X loading solution (2xTBE (1xTBE is 0.09 M Tris-borate, pH 8.0, and 0.002 M EDTA), 40% 695

sucrose, and 0.1% bromphenol blue) was added before loading on 15% urea-PAGE in 1xTBE. The 696

RNA samples were electrophoresed at 2.5 V/cm and then blotted to a Hybond N+ membrane 697

(Amersham Biosciences), and UV cross-linked. The membrane was prehybridized in 50% 698


https://doi.org/10.1101/2020.01.07.897710

23

formamide, 7% SDS, 50 mM NaHPO4/NaH2PO4, pH 7.0, 0.3 M NaCl, 5X Denhardt’s solution 699

(1X Denhardt’s solution is 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, and 0.02% bovine serum 700

albumin), and 100 mg/ml sheared, denatured salmon sperm DNA at 37 °C for at least 3 h. The 701

probe was prepared by labeling the small fragment of PiSulT gene using [γ-32P] ATP and 702

polynucleotide kinase as per the instructions manual (Molecular Labeling and Detection, 703

Fermentas) and was added to the pre-hybridization solution. The hybridization was performed at 704

37 °C overnight, and the membrane was subsequently washed at 37 °C in 2X SSC (1X SSC is 0.15 705

M NaCl and 0.015 M sodium citrate) and 0.2% SDS for 15 min twice. Final washing was given 706

only with 2X SSC at room temperature for 10 min and autoradiography was done. DNA 707

oligonucleotides 16 and 22 nucleotides (nt) were used as molecular size markers for siRNA 708

analysis. 709

710

Bi-compartment assay: 711

712

A 6-cm Petri dish (compartment 2) placed inside a 15-cm Petri dish (compartment 1) for 713

setup bi-compartment experiment to make a physical barrier between both compartments. S. indica 714

was grown in compartment 2. Surface-sterilized maize seeds were placed in compartment 1 to 715

grow plants. The leafy shoots protruded through a groove cut in the lid of each dish and were fixed 716

in one position by wrapping a sterile non-absorbent cotton wool around the portion of the 717

subtending rhizome as it passed through the groove. In both the compartments co-cultivation MN 718

media was used. Three sets were prepared for the experiment (a) maize plants colonized with WT 719

S. indica (b) maize plants colonized with S. indica-KD-PiSulT, and (c) maize plants are grown 720

alone without S. indica. In all the cases 10 µM sulphate concentration was used in compartment 1 721

as well as in compartment 2. For sets “a” and “b” to establish colonization between maize roots 722

(compartment 1) and S. indica (compartment 2) a connective bridge was made by placing a 4 to 5 723

cm long agar strip so that S. indica can cross into the compartment 1. In the case of set “c”, a 724

connecting bridge was also made to check any transfer of radioactive sulphate from compartment 725

2 to 1 due to diffusion, and this set was used as a control. As the colonization develops extraradical 726

hyphae proliferate in the medium surrounding the roots in compartment 1 where they ramify and 727

later sporulate. After colonization establishment, the MN media in compartment 2 of all three sets 728

replaced with fresh MN media containing 100 µM Sulphate and 1 µM of 35S (specific activity, 200 729

mCi/mmol). Radioactivity determines in all three sets by autoradiography, and the amount of 35S 730


https://doi.org/10.1101/2020.01.07.897710

24

incorporated measured by a liquid scintillation analyzer (Packard). The experiment was conducted 731

three times independently. 732

733

Spatial expression analysis of PiSulT: 734

735

To determine the PiSulT expression in external hyphae and in internal hyphae of the S. 736

indica colonized maize plant root, relative quantitative RT-PCR was performed as described 737

(Yadav, 2010). In brief, external hyphae projecting out from the surface of the colonized root were 738

collected by forceps. Approximately 2 mg of hyphae were collected per sample. In the case of 739

internal hyphae sample collection, first, external hyphae were removed using forceps and or 740

brushed off with a paint brush. Small pieces (5–10 mm) of colonized root were collected. 741

Colonization was also confirmed in these collected root pieces as described previously (Narayan et 742

al., 2017). RNA was isolated from these two samples, and cDNA was synthesized with the 743

Superscript cDNA synthesis kit (Clontech) and used as a template for PCR with gene-specific 744

primers for PiSulT and PiTef gene (control) (Supplemental Table 7). Quantitative RT-PCR was 745

performed as described in the previous section. 746

747

Plant metabolite measurements: 748

749

To determine total sulfur contents, plants were harvested and dried in an oven at 1500C and 750

crushed to make the fine powder. This powder was used for Energy Dispersive X-ray Fluorescence 751

(ED-XRF) (PANalytical Epsilon 5) for measuring the total sulphate contents (per gram of dry 752

weight). For sulphate ions measurement, 50 mg of frozen plant material was homogenized in 1 ml 753

of deionized water containing 20 mg of polyvinylpolypyrrolidone. The sample was incubated with 754

constant shaking at 4°C for 2h, at 95°C for 15 min and centrifuged at 14000 g for 20 min. 200 µl 755

of supernatant was used to analyze by high-performance liquid chromatography (HPLC) (Agilent 756

Technologies, Santa Clara, CA, USA, 1260 series) as described (Sieh et al 2013). Glutathione was 757

extracted from the maize plant tissue by grinding 100 mg of frozen material in 1 mL of 0.1M HCl. 758

The extract was centrifuged at 20,000 g for 10 min to remove cell debris. The supernatant was 759

used to measure the total glutathione content after reduction with dithiothreitol and subjected to 760

HPLC analysis using the monobromobimane derivatization (Sieh et al. 2013). 761

762


https://doi.org/10.1101/2020.01.07.897710

25

Expression analysis of sulphate assimilation pathway genes of S. indica and maize plant: 763

764

To investigate the relative expression of sulphate assimilation related genes of S. indica 765

during axenic and colonization with the host plant, total RNA was extracted from the non-766

colonized and colonized S. indica and maize plants under sulphate-limited and sulphate-rich 767

conditions. S. indica mycelia were grown in KF media for 7 days in high sulphate condition (10 768

mM) and filtered in minimal media containing low sulphate (LS = 10 μM) and high sulphate (HS 769

= 10 mM) further grown for 7 days. Different sulphate concentrations were given to acclimatized 770

S. indica by adding 10 μM (LS) and 10 mM (HS) in MN medium (0.4 mM NaCl, 2.0 mM 771

KH2PO4, 0.3 mM (NH4)2HPO4, 0.6 mM CaCl2, 0.6 mM MgSO4, 3.6 mM FeCl3, 0.2 mM Thiamine 772

hydrochloride, 0.1% (w/v) Trypticase peptone, 1 % (w/v) Glucose, 5 % (w/v) Malt extract, 2 773

mM KCl, 1 mM H3BO3, 0.22 mM MnSO4.H2O, 0.08 mM ZnSO4, 0.021 mM CuSO4, pH 5.8). 774

After 1 week of different sulphate concentration supply, the fungus was immediately harvested 775

and frozen in liquid nitrogen. In the case of colonization, maize plants were submerged in MN 776

media supplemented with low sulphate (LS = 10 μM) and high sulphate (HS = 10 mM) for 1 week 777

and the samples were frozen immediately, and the total RNA was isolated. Sulphate assimilation 778

pathway-related genes of S. indica (Supplemental Table 4) and maize plant (Supplemental 779

Table 5) were explored by BLASTp search. Two-step Real time-PCR protocol was used in 780

different conditions. Real time-PCR reactions were performed on an ABI 7500 Fast sequence 781

detection system (Applied Biosystems, Life Technologies, USA). The following cycles were used 782

in the ABI 7500 Fast system (96 wells plates): pre-incubation at 95°C for 5 min, denaturation 783

94°C for 10 sec (4.8C/s), annealing at 60°C for 10 sec (2.5°C/s), extension at 72°C for 10 sec 784

(4.8°C/s), 40 cycles of amplification and final extension at 72°C for 3 min. The Ct values were 785

automatically calculated, the transcript levels were normalized against PiTef expression in the case 786

of S. indica (Kumar et al., 2009) and against Actin in the case of maize and the fold change was 787

calculated based on the non-treated control. The fold change values were calculated using the 788

expression, where ∆∆CT represents ∆CT condition of interest gene- ∆CT control gene. The fold 789

expression was calculated according to the 2-∆∆CT method mentioned elsewhere (Livak and 790

Schmittgen, 2001). 791

792

793

794


https://doi.org/10.1101/2020.01.07.897710

26

Statistical Methods: 795

796

The statistical analyses were performed with Microsoft Excel 2010 and GraphPad Prism 8. 797

The significance of the study was calculated using one-way ANOVA. 798

799

Accession Numbers: 800

801

Sequence data of S. indica sulphate transporter (PiSulT) can be found in the Gene bank 802

Database (https://www.ncbi.nlm.nih.gov/genbank/) under the following accession number: 803

MG816118.1 804

805

Supplemental Data: 806

Supplemental Table 1. List of domain hits. 807

Supplemental Table 2. Homology between highly similar fungus species with putative PiSulT 808

Supplemental Table 3. Homology of PiSulT with another organism. 809

Supplemental Table 4. Fold change of sulfur assimilation pathway genes of S. indica. 810

Supplemental Table 5. Fold change of sulfur assimilation pathway genes of the maize plant. 811

Supplemental Table 6. List of strains, and plasmids used in this study. 812

Supplemental Table 7. List of oligonucleotides used in this study. 813

Supplemental Figure 1. PiSulT ORF sequence and codon wise predicted amino acid sequences. 814

Supplemental Figure 2. PiSulT ORF sequence. 815

Supplemental Figure 3. Schematic representation of genomic orientation, ORF and protein 816

translation of putative PiSulT gene. 817

Supplemental Figure 4. Multiple sequence alignment analysis. 818

Supplemental Figure 5. Multiple sequence alignment analysis. 819

Supplemental Figure 6. Colonization pattern of S. indica in maize roots. 820

Supplemental Figure 7. Chromate toxicity in the presence of different concentrations of sulphate. 821


https://doi.org/10.1101/2020.01.07.897710

27

Supplemental Figure 8. Effect of different concentrations of chromate on sulphate transport in WT 822

complemented and mutant of sulphate transporter. 823

Supplemental Figure 9. pRNAi yeast shuttle vector and RNAi insert map. 824

Supplemental Figure 10. Knockdown of PiSulT gene of S. indica. 825

Supplemental Figure 11. Growth analysis of pRNAi-PiSulT transformed S. indica. 826

Supplemental Figure 12. Bi-compartment Petri dish culture system to study the transport of 827

radiolabeled (35S) sodium sulphate to maize plants via S. indica. 828

Supplemental Figure 13. Relative expression analysis of putative sulphate assimilation pathway 829

genes of WT-S. indica grew axenically under low sulphate. 830


genes of KD-S. indica grew axenically under low and high sulphate conditions. 832


genes of WT-S. indica during the colonized stage with maize plants under low and high sulphate 834

conditions. 835


genes of KD-S. indica during the colonized stage with maize plants under low and high sulphate 837

conditions. 838

Supplemental Figure 17. Expression analysis of sulphate assimilation genes of maize plant during 839

WT-S. indica colonized stage under low and high sulphate conditions. 840

Supplemental Figure 18. Expression analysis of sulphate assimilation genes of maize plant during 841

KD-PiSulT-S. indica colonized stage under low and high sulphate conditions. 842

843

844

AUTHOR CONTRIBUTIONS 845

846

AKJ has initiated the project. OPN and NV have performed the experiments. OPN, AKJ, 847

AJ, and MD have designed the experiments. Chemicals were provided by AKJ and MD. The 848

project was supervised by AKJ and MD. MS is written by OPN and AKJ. 849

850

The authors declare no conflict of interest 851

852

853


https://doi.org/10.1101/2020.01.07.897710

28

ACKNOWLEDGMENTS 854

855

We are very thankful to Prof. Johan M. Thevelein, Laboratory of Molecular Cell Biology, 856

Institute of Botany and Microbiology, KU Leuven, Kasteelpark Leuven-Heverlee, Flanders, 857

Belgium, for providing yeast sulphate transporter mutant (HK14) and WT strain (BY4742) for 858

study. OPN is grateful to the Indian Council of Medical Research (ICMR), Government of India 859

for its financial support. NV is thankful to Jawaharlal Nehru University for providing a research 860

fellowship. We are also very thankful to Prof. Alga Zuccaro, Institute for Genetics, Cologne 861

Biocenter University of Cologne, Germany, for providing the pRNAi vector. AKJ and MD are 862

thankful to Jawaharlal Nehru University for providing DST-PURSE-II, UPOE-II, and UGC-863

Resource NET-working grant. 864

865

866

867

868

869

870

871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

886

887

888

889


https://doi.org/10.1101/2020.01.07.897710

29

890

891

892

Table 1. Summary of amino acid identity (%) between S. indica PiSulT and other fungal, 893

plant, animal, insect and prokaryotic sulphate transporters. 894

895

896

Organism Description Sulphate Transporter

(no. of amino acids)

Gene BankTM

Accession No.

Identity with

PiSulT %

S. indica (Fungus) PiSulT (763) CCA67103.1 100

S. cerevisiae (Fungus) Sul1 (859) AJP84964.1 42

H. sapiens (Mammal) SLC26A11 (606) NP 001159819.1

33

D. melanogaster (Insect) Esp (623) AAD53951.1

30

A. thaliana (Plant) SULTR1 (649)

OAO99615.1 29

E. coli (Bacteria) YchM (550)

SCQ14120.1 27

897

898

899

900

901

902

903

904

905

906

907

908

909

910

911

912

913


http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Get&ALIGNMENTS=100&ALIGNMENT_VIEW=Pairwise&CDD_RID=data_cache_seq%3A353235085&CDD_SEARCH_STATE=4&DATABASE_SORT=0&DESCRIPTIONS=100&DYNAMIC_FORMAT=on&FIRST_QUERY_NUM=0&FORMAT_OBJECT=Alignment&FORMAT_PAGE_TARGET&FORMAT_TYPE=HTML&GET_SEQUENCE=yes&I_THRESH&LINE_LENGTH=60&MASK_CHAR=2&MASK_COLOR=1&NEW_VIEW=yes&NUM_OVERVIEW=100&OLD_BLAST=false&PAGE=Proteins&QUERY_INDEX=0&QUERY_NUMBER=0&RESULTS_PAGE_TARGET&RID=1AMVPCBT014&SHOW_LINKOUT=yes&SHOW_OVERVIEW=yes&STEP_NUMBER&WORD_SIZE=6&DISPLAY_SORT=3&HSP_SORT=3

http://www.ncbi.nlm.nih.gov/protein/353235085?report=genbank&log%24=prottop&blast_rank=1&RID=1AMVPCBT014

https://doi.org/10.1101/2020.01.07.897710

30

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Figure 1. Phylogenetic tree of sulphate transporters. Phylogenetic tree of PiSulT with

sulphate transporters of plants, animals, fungi, and prokaryotes. The tree was generated by

MEGA-X software using Muscle for the alignment and the neighbor-joining method for the

construction of the phylogeny. The bootstrap test was performed using 1000 replicates. The

branch lengths are proportional to the phylogenetic distance. The distance scale showing

genetic variation for the length of the scale (filled shape indicates S. indica). Value 0.1

showing distance scale, which represents the number of differences between sequences (e.g.

0.1 means 10 % differences between two sequences).


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Figure 2. Expression pattern of PiSulT in response to different concentrations of

sulphate: A. qRT-PCR of PiSulT gene isolated from S. indica grown in MN media

containing the indicated different sulphate concentrations at 1, 5, 10, and 15 days. PiTef gene

was used as a reference gene. B. Semi-quantitative RT-PCR of PiSulT gene isolated from S.

indica under similar conditions. Colonization of S. indica with maize root: C. Trypan blue

staining of maize plant roots showing the presence of intracellular chlamydospores of S.

indica in the cortical cells at 5, 10, 15 and 20 days of colonization (+PI) (spores are shown in

blue color) with the maize plant grown in sterile soil supplemented with the Hogland

solution. 5 and 20-days plants without S. indica (-PI) were used as a negative control. D.

Expression of PiSulT during colonization with maize plant: qRT-PCR showing the

amplification of PiSulT transcripts from colonized maize plant roots at 5, 10, 15, and 20 days

to confirm the presence of S. indica into plant root tissue. PiTef was used as a reference gene.


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Figure 3. A. Drop test analysis for yeast sulphate transporter mutant HK 14 complementation. (i) Yeast cells were grown at 30ºC on SD media containing 0.1 mM of sulphate as a sole source of sulfur in the presence of 2% glucose (non-inducing condition) and 2% galactose (inducing condition) as sole carbon source separately. Cells were suspended in sterile distilled water and cell density was adjusted to A600= 0.1, followed by serial dilutions of 1/10 (from left to right). Upper lane showing growth pattern of WT parent strain (BY4742) (used as a positive control), middle lane showing growth pattern of mutant transformed with empty vector (used as a negative control) and lower lane is showing growth pattern of mutant HK14 complemented with PiSulT (ii) Growth pattern (using streak method) of mutant HK14 complemented with PiSulT, WT and mutant transformed with the empty vector were shown under similar conditions mentioned above. B. Growth pattern study: (i)


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2

WT yeast (BY4742) (ii) mutant HK 14 transformed with PiSulT. In both, cases cells were starved for sulfur and then transferred to SD medium containing a different concentration of sulphate (as indicated) as the sole source of sulfur (iii) WT, mutant HK14 complemented with PiSulT and mutant HK 14 transformed with only vector (empty vector). In this case, cells were starved for sulfur and then transferred to medium containing 3mM concentration of sulphate as the sole source of sulfur.


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Figure 4. Functional characterization of PiSulT gene using sulphate transporter mutant

HK14 (sul1Δ&sul2Δ): A. Sulphate uptake by yeast HK14 mutant cells transformed with

empty vector (black), with PiSulT (vertical lines) and WT parent strain, BY4742, (crossed

line) (used as a positive control). Means and standard errors of the means of the three

replicate determinations consisting of three measurements B. Kinetics analysis of S35

uptake

in a yeast mutant HK14 complemented with PiSulT and WT (BY4742). Nonlinear regression

of sulphate uptake rate was measured after 4 minutes of time when cells transferred to media

containing S35

labeled Na2SO4 in the presence of galactose at pH 5. The graph was calculated

using Graph Pad Prism 6 software. The analysis was performed in triplicates and significance

has been calculated using one-way ANOVA C. Determination of the optimum pH for

sulphate uptake assay by PiSulT. The readings are relative to the negative control (HK 14

transformed with an empty vector).


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Figure 5. Transport of sulphate to maize plants by S. indica carried out in the bi-

compartment Petri dish culture system. Radioactivity incorporated into plants was

demonstrated by autoradiography. Radioactivity count intensities are shown in the false color

code (vertical bar, low to high). Panels. A. Maize plants were colonized with WT S. indica

designated as WT, B. Maize plants were colonized with KD-PiSulT-S. indica designated as

KD; C. Maize plants were grown alone without S. indica designated as C. (i), whole maize

plant before autoradiography; (ii), false-color autoradiograph of the maize plant obtained

after 12 h of exposure of the maize plant; and (iii), microscopic view of a sample of plant root

showing colonization and non-colonization. D. The content of transferred sulphate: Amount

of 35

S transferred to the maize plant components by S. indica. Radioactivity was measured

three times independently (the number of transformants used was n=3). The mean S.D. of

three independent measurements is shown. * indicate a significant difference (p<0.01). E. Spatial expression of PiSulT during colonized condition. Expression of PiSulT in the external (EH)

and internal hyphae (IH) of S. indica during colonization with maize plant. Relative fold change in IH

was compared with EH. Tef gene was used as an internal control. The values obtained for PiSulT

expression for EH and IH were 1 and 0.449-fold respectively. The means S.D. of three independent

determinations is presented. * indicate significant difference from EH (p<0.01).


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Figure 6. The impact of PiSulT on plant health and development and on sulphate nutritional

enrichment. A. Maize plant colonized with WT S. indica showing improved growth as compared to

maize plants colonized with KD-PiSulT-S. indica and maize plants without S. indica. B. Biomass

study of maize plants colonized with WT S. indica showing improved growth as compared to maize

plants colonized with KD-PiSulT-S. indica and maize plants without S. indica (control). C. Sulphate

content. D. The impact of S. indica on host plant under high or low sulphate concentration. Total

biomass (fresh weight) of maize plants grown under low (10uM) and high (10mM) sulphate

conditions with or without S. indica colonization. E. Sulphate content. (-S= low sulphate, +S= high

sulphate +Pi= plant colonized with S. indica, –Pi= non-colonized plant). Different asterisk indicates

significant differences, * significant (p<0.05), **(p<0.01), ***highly significant (p<0.001). Tukey

test was used to check the significance.


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Figure 7. The impact of PiSulT on the improvement of plant metabolite and sulphate

nutrition. Measurement of glutathione (GSH) content (A) and sulphate content (B) in maize

plants grown under low (10uM) and high (10mM) sulphate conditions with or without S.

indica colonization (-S= low sulphate, +S= high sulphate +Pi= plant colonized with S. indica,

–Pi= non-colonized plant). Different asterisk indicates significant differences, * significant

(p<0.05), **(p<0.01), ***highly significant (p<0.001). Tukey test was used to check the

significance. Data were analyzed three independent times in triplicates.


https://doi.org/10.1101/2020.01.07.897710

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https://doi.org/10.1101/2020.01.07.897710

Role of Sulphate Transporter (PiSulT) of Endophytic Fungus … · 2020. 1. 8. · 1 1 Role of Sulphate Transporter (PiSulT) of Endophytic Fungus Serendipita indica in Plant 2 Growth

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