PiHOG1, a stress regulator MAP kinase from the root endophyte fungus Piriformospora indica, confers salinity stress tolerance in rice plants

In this study, yeast HOG1 homologue from the root endophyte Piriformospora indica (PiHOG1) was isolated and functionally characterized. Functional expression of PiHOG1 in S. cerevisiae ∆hog1 mutant restored osmotolerance under high osmotic stress. Knockdown (KD) transformants of PiHOG1 generated by RNA interference in P. indica showed that genes for the HOG pathway, osmoresponse and salinity tolerance were less stimulated in KD-PiHOG1 compared to the wild-type under salinity stress. Furthermore, KD lines are impaired in the colonization of rice roots under salinity stress of 200 mM NaCl, and the biomass of the host plants, their shoot and root lengths, root number, photosynthetic pigment and proline contents were reduced as compared to rice plants colonized by WT P. indica. Therefore, PiHOG1 is critical for root colonisation, salinity tolerance and the performance of the host plant under salinity stress. Moreover, downregulation of PiHOG1 resulted not only in reduced and delayed phosphorylation of the remaining PiHOG1 protein in colonized salinity-stressed rice roots, but also in the downregulation of the upstream MAP kinase genes PiPBS2 and PiSSK2 involved in salinity tolerance signalling in the fungus. Our data demonstrate that PiHOG1 is not only involved in the salinity response of P. indica, but also helping host plant to overcome salinity stress.

Homology and phylogenetic analysis. CLUSTALW analysis revealed that PiHOG1 contains conserved functional domains and sites characteristic of the HOG1 protein. In case of PiHOG1 MAP kinase, a TGY phosphorylation site was observed at amino acid position 170-172 (green-shaded, Fig. 1A) as well as a C-terminal common docking motif was also present which contains two aspartic acid residues (D) at position 303 and 306 (rectangle shape, Fig. 1A), and PBS2 binding domain-2 (yellow shaded region, Fig. 1A). PiHOG1 showed highest similarity to HOG1 from root pathogenic basidiomycete fungus Heterobasidion annosum. InterProscan and conserved domain analysis show the presence of a protein kinase catalytic domain, a serine/threonine-dual specificity protein kinase catalytic domain, a MAP kinase conserved site, a protein kinase ATP binding site, a C-terminal common docking motif, a P38 MAP kinase and tyrosine kinase domain. BLASTx and BLASTp suggested that PiHOG1 is a member of salt-activated MAP kinases (SAMAPKs).
An amino acid level similarity of PiHOG1 with other different salt-activated or -induced MAP kinases is shown in Table S1. Highest similarities of PiHOG1 were observed with plant interacting fungi as compared to more primitive fungi. Interestingly, the similarities of PiHOG1 were higher to filamentous (Schizosaccharomyces) Sty1 MAP kinase compared to fission yeast S. cerevisiae HOG1 (Table S1; Fig. 1A,B). Phylogenetic analysis suggested that putative PiHOG1 falls closely into basidiomycete fungi family and also with the plant interacting fungi from Ascomycota (Fig. S3).
PiHOG1 functionally complements ∆hog1 mutant of S. cerevisae. We found that, when higher than 0.25 M monovalent salts (NaCl and KCl) treatment was given, ∆hog1 mutant could not survive, however PiHOG1 complemented yeast mutant was found to be survived as good as WT. A comparable survival was observed for the PiHOG1 complemented ∆ hog1 mutant up to 1.5 M monovalent osmostress ( Fig. 2A). In case of divalent salts (CaCl 2 and MgCl 2 ), growth of PiHOG1 complemented ∆ hog1 mutant was found to be comparable to that of WT. However, all yeast strains were incapable to survive on more than 500 mM of divalent salt (Fig. 2B). Furthermore, PiHOG1 complemented ∆ hog1 mutant yeast survived up to 2 M sorbitol osmostress and growth was found to be similar to that of WT (Fig. 2C). PiHOG1 complementation also restored tolerance against oxidative stress imposed by supplementing 5 mM H 2 O 2 as we observed a growth in complemented mutant (Fig. 2D).
As P. indica is a native of Thar Desert which is a harsh environment having high temperature and drought condition. To adapt in such situations, HOG1 might play role in heat stress tolerance. Therefore complemented hog1 mutant was also tested under heat stress. We found that at 40 °C, PiHOG1 complemented yeast hog1 mutant also restored heat tolerance comparable to WT (Fig. 2E).
Glycerol accumulation, morphology and growth of PiHOG1 complemented yeast mutant under salinity stress condition. We found that upon osmotic stress (0.5 M NaCl), PiHOG1 complemented hog1 mutant showed increased glycerol level comparable to WT. In response to osmostress the glycerol concentration was higher in PiHOG1 complemented mutant as compared to ∆hog1 mutant (Fig. 3A). In yeast, HOG1 plays important role in growth and morphology maintenance under osmo-stress condition of 1 M NaCl. In case of ∆hog1 mutant and empty vector control ∆hog1 mutant cells, we observed aberrant cell shape i.e., large multinucleated cells with multiple elongated buds when osmotic stress was given however, no such defect was observed in case of WT (Fig. 3C). Further, we found that PiHOG1 complemented mutant was able to restore normal morphology comparable to WT as cells were found to be morphologically similar (Fig. 3C).
It was found that growth of ∆hog1 mutant was very slow; however in case of PiHOG1 complemented mutant growth was comparable with WT. It shows that PiHOG1 was also restoring slow growth of yeast mutant under osmostress condition {Fig. 3B(b-d)} whereas in SD control medium all yeast strain were found to grow in almost similar fashion {Fig. 3B(a)}.

Expression analysis of HOG pathway and salinity tolerance genes in axenically grown P. indica.
Expression of salinity tolerance genes (Table S2A,B) in WT P. indica and KD-PiHOG1 P. indica was analysed in normal and high salinity conditions by semi quantitative and real time-PCR. In case of WT P. indica (exposed to 0.5 M NaCl for 1 h), out of 11 selected HOG pathway homologue genes 10 genes (Table S2A; PiHOG1, PiPBS2, PiSSK2, PiPFK26, PiHSP78, PiGRE2, PiGPD1, PiSTL1, PiENA1 and PiPMC1) were found to be upregulated and 1 was found to be downregulation as compared to the P. indica grown under normal condition (MN media, no salt). PiHOG1 and PiENA1 were up-regulated 30 and 46.5-folds, respectively (Fig. S5A). Out of three ATPase ion channels (PiENA1, PiPMR1 and PiPMC1) Na + -K + ATPase PiENA1 was highly upregulated. PiPMR1 was found to be downregulated (Figs S5A and S6). In case of KD-PiHOG1 P. indica, out of 11 genes only 3 genes were found to be upregulated upon osmostress shock as compared to the non-salt treated KD-PiHOG1 P. indica. In case of Schizosaccharomyces pombe Sty1 MAP kinase (NP_592843.1) and S. cerevisiae HOG1 (U53878) were aligned with the CLUSTALW software. The serine/threonine protein kinase catalytic domain is shaded by gray (25%) in which the conserved TGY phosphorylation motif is distinguished by green shade. The C-terminal common docking (CD) motif is shown in rectangle shape in which the conserved hydrophobic amino acids tyrosine (Y) and histidine (H) are underlined and conserved acidic aspartic acids (D) are dark yellow shaded. PBS2 binding domain-2 is shaded in yellow color. [*, perfectly conserved residues, :, very similar residues, •, similar residues]. (B) Phylogenetic tree with branch lengths: The tree was constructed by using different stress-activated MAP kinase/HOG1/P38 amino acid sequences. Member of different groups were marked with different shape i.e. Δ : insects, □ : mammals, ◊ : fungi and ○ : plants. PiHOG1 protein is marked with filled shape to display its position.
Scientific RepoRts | 6:36765 | DOI: 10.1038/srep36765 KD-PiHOG1 P. indica only PiPMC1 and PiPMR1 (1.8 and 1.6 fold respectively) were found to be upregulated however rest of the genes were found to be downregulated, as compared to the non-salinity treated KD-PiHOG1 P. indica (Figs S5B and S6).

PiHOG1 knockdown affects colonization of P. indica, plant growth and development. P. indica
colonization of rice plant roots was checked after 15 days post inoculation (dpi). In normal condition, P. indica transformed with KD-PiHOG1 showed 55% colonization as compared to 75% observed in case of WT P. indica. However, under salinity stress condition, 70% colonization was observed in case of WT P. indica as compared to the 40% colonization observed in case of KD-PiHOG1-P. indica at 15 dpi ( Fig. 4A-D). Under similar condition, in case of KD-PiHOG-P. indica chlamydospores were observed in clumps rather than in chain form (Fig. 4B) as compared to WT P. indica (Fig. 4A). Moreover, the spores were more in number on root surface in case of KD-PiHOG1-P. indica inoculated roots as compared to WT (Fig. 4A,B).  PiHOG1 knockdown affects photosynthetic pigments and proline content. A major response of salinity stress in plants is the degradation of photosynthetic pigments which is caused by chlorosis, reduced photosynthesis and oxidative damage. As a result, plants become brownish, have stunted growth and reduced weight. The photosynthetic pigments (Chl a, Chl b and carotenoid) were found to be decreased in KD-PiHOG1 P. indica inoculated plants as compared to the WT P. indica-inoculated plant {Fig. 5B(f-h)}.
In plants proline accumulation is considered as immediate response to combat osmostress. It was observed that the proline content increased significantly in P. indica-inoculated rice plants as compared to the non-inoculated plants when 200 mM NaCl treatment was given {Fig. 5B(i)}. Interestingly, enhanced proline content was observed in case of WT and KD-PiHOG1 P. indica -inoculated rice plants which are not exposed to salinity stress as compared to the non-inoculated plants. Under osmostress condition, KD-PiHOG1 P. indica-colonized plants were having less proline content than WT P. indica-colonized plants {Fig. 5B(i)}.
Expression analysis of HOG pathway and salinity tolerance genes of P. indica upon osmostress during colonized stage. Most of the HOG pathway genes were found to be up-regulated upon osmostress except PiPFK26 and PiPMC1. We found that PiHOG1, PiENA1, PiPBS2, PiSTL1, PiGPD and PiSSK2 were induced up to 8-folds in case of WT P. indica colonized with the rice plant and treated with the 0.5 M NaCl as compared to the WT P. indica colonized with the rice plants under non-salinity stress ( Fig. S9A; Table S2).
Moreover, calcium channel PMR1 was the only gene which was observed to be 1.8 fold up-regulated in case of KD-PiHOG1 P. indica colonized with the rice plant as compared to the WT P. indica colonized with the rice plants under salinity stress. However a very week i.e., 1.04-fold up-regulation of PiHOG1 was also observed under similar condition ( Fig. S9A; Table S2). These results clearly show the important role of PiHOG1 as a central player in regulating HOG pathway genes for survival of P. indica even in colonized stage which is almost reversed upon PiHOG1 knock down. The activity of HOG pathway may be necessary for survival during non-stress as well as stress condition during colonization. Although overall activity of HOG pathway suppressed in non-stress colonized stage, yet this level might be necessary for strategic survival.
Further, the salinity tolerance conferring genes 22 of P. indica were also analyzed during colonized condition. Out of 20 selected genes, only two genes i.e. Sphingolipid C9-methyltransferase-like protein (PiSLC9M) and Cytochrome P450-like protein (PiCP450) were found 1.6 and 4.4 folds up-regulated respectively in case of WT P. indica colonized with the rice plant (treated with the 0.5 M NaCl) as compared to the non-salinity treated P. indica colonized with the rice plant. We observed that when P. indica was grown axenically under salinity stress conditions 12 genes were found to be upregulated (Fig. S7A, Table S2) whereas in colonized stage only 2 of them were found to be up-regulated ( Fig. S9B; Table S2). This suggests that these 12 genes might play role in salinity tolerance axenically rather than in colonized stage. During colonized stage PiSLC9M gene was found to be up-regulated but was found to be down-regulated during axenic osmostress condition (Fig. S7A). This suggests the unique role of this gene in colonized stage. In case of KD-PiHOG1 P. indica colonized with plants, 5 salinity tolerance genes were found to be upregulated and rest of the genes were found to be downregulated ( Fig. S9B; Table S2). PiSLC9M, (1.15-fold), polyubiquitin-like protein (PiPULP, 1.3-fold), 27S glyceraldehyde 3-phosphate dehydrogenase (Pi27SGDP, 2.27-folds), BCL-2 associated athanogene 3-like protein (PiBA3LP, 1.16-fold) and cytochrome P459 (PiCP459, 1.36-fold) were found to be upregulated in KD-PiHOG1 P. indica-colonized with plant (treated with the 0.5 M NaCl) as compared to WT P. indica-colonized with the plant ( Fig. S9B; Table S2).

Phosphorylation of PiHOG1 during interaction of P. indica and rice plant. Phosphorylation is
the mode of HOG1 activation in yeast. PiHOG1 get activated and phosphorylation was observed when 0.5 M NaCl salinity stress shock was applied (Fig. 6A). During colonized state in case of WT P. indica PiHOG1 gets phosphorylated even during non-salinity condition at 0 min. Phosphorylation of PiHOG1 was found to be more at 30 min. However in case of KD-PiHOG1 P. indica PiHOG1 phosphorylation did not occur in non-salinity condition at 0 min as compared to WT P. indica. Also less phosphorylation was observed from 15 mins to 60 min as compared to WT P. indica (Fig. 6A). PiHOG1 was not found upregulated in rice colonized KD-PiHOG1 P. indica as compared to WT P. indica upon salinity stress. In our study, PiHOG1 knockdown results in downregulation of upstream molecules MAP kinase kinase kinase PiSSK2 (0.9-fold) and MAP kinase kinase PiPBS2 (0.76-fold). During salinity stress in rice colonized WT P. indica, PiSSK2 (2.7-folds) and PiPBS2 (5.5-folds) were found to be upregulated (Fig. 6B).

Discussion
The mutualistic root endophyte fungus P. indica seems to evolve in harsh environmental conditions as it is a native to Thar desert of Rajasthan, India which is an extreme drought habitat 1 . Endophytic association of P. indica has been proven as beneficial tool for host plant to survive under abiotic stresses such as salinity and drought 5,8,9,23 . We found that P. indica can tolerate up to 250 mM NaCl. Other fungi like C. albicans, Heterobasidion annosum, Botrytis cinerea, and Cochliobolus heterostrophus also show a higher osmotolerance level when exposed to NaCl and thus support our data [24][25][26][27] . We found that divalent salts severely retard the growth and found to be more toxic to the P. indica as compared to monovalent salts. This finding can be explained as divalent salts generate more osmotic pressure as compared to the monovalent salts. Further divalent salts have been reported to have higher toxicity which results in retard growth 28,29 .
It is known that fungi have quickly responding MAP kinase signalling pathways 30 . The MAP kinase osmoregulatory HOG response pathway is conserved in all eukaryotes (except plants) including fungi, mammals and Phosphorylation of PiHOG1 in extracts from symbiotic rice roots colonized by eihter WT or KD-PiHOG1 P. indica was measured 0-60 min after the application of 0.5 M NaCl. Coomassie Blue (CB) stain was used as control for equal protein loading which was measured using Bradford assay. (B) Expression of HOG MAP kinase cascade genes of WT P. indica and KD-PiHOG1 P. indica exposed to 0.5 M NaCl for 1 hr during colonizing stage with rice plant: The transcript levels of putative HOG MAP kinase cascade genes were quantified. Fold change variation of the genes compared to the non-treated control was calculated and PiTef as endogenous reference was used. Gene expression in the WT P. indica under non-salt condition was set to 1. (C) Proposed osmoregulatory and osmodaptation pathway (putative HOG pathway) in the root endophyte P. indica: The picture of single P. indica cell is showing the perception of the stress signal via putative or unknown osmosensors (such as putative SLN1 and SHO1) which may be transduced to putative MAP kinase cascade through phosphorelay signal transduction. As a result, MAP kinase PiHOG1 might get phosphorylated at TGY motif which might activate the putative osmoresponsive transcription factors (ORTFs) of osmoresponsive genes (ORGs) and initiate transcription of ORGs to perform various responses related to stress defence, survival and homeostasis condition. Additionally, putative HOG pathway may also affect host interaction related function and morphology of fungus during host colonized stage. insects to activate responses to different stress signals 12,31 . We found that PiHOG1 not only have similarity with other known HOG1 homologs from closely related host interacting fungi but also exhibited similarity with mammals. Our phylogenetic analysis of PiHOG1 showed nearer neighbourhood to stress-activated MAP kinases or with HOG1 from plant interacting fungi than to other fungal species. In case of EhHOG1 from Dead Sea-isolated fungus Eurotium herbariorum 32 , growth and aberrant morphology of hog1 mutant was restored under high osmotic stress condition which is comparable with PiHOG1 complemented hog1 mutant thus support our data. In yeast, the glycerol accumulation is the resulting response of HOG1 protein activation under osmostress condition 15 . In the present study also glycerol accumulation was found to be restored in PiHOG1 complemented yeast mutant and it was found to be 3-folds higher as compared to hog1 mutant exposed to salinity stress (NaCl). PiHOG1 restored growth, morphology, heat tolerance and oxidative stress of mutant yeast. We found that osmotolerance capacity of KD-PiHOG1 P. indica was dramatically decreased as compared to WT P. indica. The radial growth of KD-PiHOG1 P. indica reduced up to 80% on different osmostress agents as compared to WT P. indica. Further, the growth of KD-PiHOG1 P. indica was affected severely on divalent salts as compared to the monovalent salts.
During salinity stress, role of PiHOG1 in conferring salinity tolerance to colonized plant is not known in any plant-fungal symbiotic interactions. HOG1 homologue of the ryegrass fungal endophyte E. festucae has been reported to play important role in conidia formation 19 . It was found that beneficial endophyte converted into pathogenic endophyte and colonization was found to be decreased when HOG1 homologue was knocked out 33 . However no such conversion from beneficial to abnormal pathogenic strain upon PiHOG1 knockdown was observed. In our study, the root percentage colonization was found to be decreased and the beneficial effects of P. indica were compromised during salinity stress condition. P. indica chlamydospores were found in clusters rather than in chain form in case of KD-PiHOG1 P. indica-colonized rice roots, mostly, they were present in epidermal region and on the root surface.
HOG pathway is not only necessary for osmotolerance regulation but also reported to play various important function in different fungi like in cell-wall integrity, conidiation, regulation of pathogenicity and alternariol biosynthesis, regulation of vegetative differentiation, virulence and appressorium formation 26,[34][35][36][37][38][39][40][41][42] . In case of P. indica, we have found PiHOG1 knockdown resulted in aberrant spore germination. Similar observations were also made in case of B. cinerea, bcsak1, which encode a mitogen-activated protein kinase (MAPK). Further, Δbcsak1 mutants were found to be significantly impaired in vegetative and pathogenic development 26 and thus support our data.
Abiotic stress tolerance conferred by P. indica to host plants has been studied extensively with barley, rice and other plants 5,9,36 . To investigate the role of PiHOG1 in protecting the plant during salinity stress, we have colonized rice plants with KD-PiHOG1 P. indica transformant and WT P. indica. We observed significant reduction in growth related parameters in case of KD-PiHOG1 P. indica-colonized plants as compared to WT P. indica colonized plants under salinity stress. Further, KD-PiHOG1 P. indica-colonized plants were showed reduced photosynthetic pigments as compared to WT P. indica colonized plants. It is worth mentioning that plants colonized with WT P. indica were found to be healthy as compared to the plants colonized with KD-PiHOG1 P. indica under salinity stress. This can be explained as more accumulation of proline was found in case of plants colonized with the WT P. indica which helps the plants to maintain osmotic balance inside the cell and protect them from toxic damage.
Additionally, HOG1 gene was found to be involved in the regulation of salinity tolerance and HOG pathway related genes under osmostress in case of S. cerevisae. Likewise, we also found that PiHOG1 playing important role in the regulation of the similar genes during the interaction of the P. indica with the rice plant under salinity stress. In the present study HOG1 pathway related genes of P. indica i.e., PiHOG1, PiPBS2, PiSSK2, PiGPD1, PiSTL1, PiPMR1, PiHSP78, PiENA1 and PiGRE2 were found to be up-regulated in case of WT P. indica colonizing rice plant as compared to KD-PiHOG1 P. indica colonized host plant plants during salinity stress. However two genes viz., PiPFK26 and PiPMC1 were found to be down-regulated under similar conditions. Similarly in case of H. annosum GPD1, HSP78, STL1 and GRE2 were found to be induced after exposure to salinity stress which supports our data. Amongst, PMC1 was found to be highly induced when the fungus was exposed to 0.2 M CaCl 2 25 . GPD1 was suggested as a key player in the response to osmotic stress in yeast 43 . STL1 which encodes an glycerol/H + symporter and regulate the glycerol accumulation under stress in S. cerevisiae was found to be up-regulated in C. albicans under osmostress condition 44,45 . C. albicans accumulates more glycerol and d-arabitol when exposed to physiological conditions related to stress and virulence in animals. It has been reported that C. albicans mutants that produce less glycerol were found to be hyper susceptible to environmental stresses and are hypovirulent in mice 46 . As glycerol work as a main protective solute and play an important role to maintain osmotic homeostasis in cell 16,47 , therefore glycerol accumulation is important for fungi to colonize and survive in mammalian hosts under less supply of nutrient, high osmolarity, temperatures, low oxygen levels and oxidative killing by host. We found that both WT yeast and complemented yeast mutant accumulates glycerol equally however in case of yeast mutant less accumulation of glycerol was observed. In case of KD-PiHOG1 P. indica transformant we have found that the genes related to the glycerol accumulation (STL1, GPD and PFK26) were found to be down-regulated therefore we hypothesize that in case of KD-PiHOG1 P. indica transformant could not resist salinity stress therefore less colonization occurred with host plant and as a result mutant fungi were failed to provide protection to the colonized plant against salinity stress. It has been reported that ENA1 is an ATPase pump which regulates Na + /K + efflux to keep the intracellular ions concentration at low level and has been reported to induce strongly in osmostress and regulated by HOG1 in yeast and found to play important role in virulence, ion homeostasis and anti-fungal resistance [48][49][50] . In our study PiENA1 was found to be down-regulated in case of KD-PiHOG1 P. indica during colonization, because of this P. indica could not have efflux out Na + due to which Na + becomes toxic to the colonized mutant fungi, hence low colonization was observed under salinity stress condition. Therefore, growth parameters, photosynthetic pigment were found to be reduced in plants colonized with the mutant fungi as compared to the WT P. indica colonized plants. In case of KD-PiHOG1 P. indica PMR1 and PMC1 was found to be down-regulated and also growth of KD-PiHOG1 P. indica was found to be retarded under multiple stresses (Fig. S4). In case of Beauveria bassiana and Hansenula polymorpha, PMR1 and PMC1 have been reported as core regulator of growth, conidiation and responses to multiple stressful stimuli 51-53 thus support our data. In case of S. cerevisiae methylglyoxal reductase GRE2 was found to be induced when osmotic shock was given. It was suggested that transcriptional induction of GRE2 to salinity stress is dependent on the HOG1, which indicate that the HOG1-mediated signalling pathway plays a key role in global gene regulation under salinity stress conditions 43 .
It has been reported that P. indica colonization with the salinity-sensitive barley plants results in an increase in the antioxidant properties and as a result plants become resistance towards salinity stress 23 . However the expression of salinity tolerance genes of P. indica was never reported. In the present study we have selected twenty salinity tolerance conferring genes 22 of P. indica and there expression was analyzed upon salinity stress and during colonization with rice plant. During non-colonizing stage, we found 12 salinity conferring genes upregulated upon osmostress in case of WT P. indica as compared to the P. indica grown under non-salt condition. Further, only glyceraldehyde 3-phosphate dehydrogenase 27S (Pi27SGDP) was found to be upregulated in KD-PiHOG1 P. indica under similar condition. However only two salinity tolerance genes i.e. PiSLC9M and PiCP450 were found to be upregulated in WT P. indica during colonization as compared to KD-PiHOG1 P. indica colonizing host plant under salinity stress. SLC9M has been reported to be involved in acid stress tolerance in gastrointestinal bacteria and in hypoxia condition in an aquatic fungus Blastocladiella emersonii 54,55 . CP450 has been reported in salinity and drought stress tolerance in case of A. thaliana and Oryza sativa 56,57 therefore support our data. In case of KD-PiHOG1 P. indica colonized with host plant, PiSLC9M, polyubiquitin-like protein (PiPULP), Pi27SGPD, BCL-2 associated athanogene 3-like protein and PiCP459 were found to be upregulated under salinity stress. It is known that in yeast, HOG1 globally affects and regulates osmoresponsive genes upon osmostress shock 58 . Our study also suggests that PiHOG1 might be playing role in regulation of these salinity tolerance genes in P. indica.
Most of the rice salinity tolerance genes were found to be downregulated even under salinity stress condition in case of rice plant colonized with the WT P. indica as compared to the rice plant colonized with the KD-PiHOG1 P. indica transformant. Further in case of KD-PiHOG1 P. indica colonized rice plant, seven genes namely OsSTK, OsLEAP, OsAP1, OsMPIX, Os40S27, OsSIP and OsSOS1 were found to be upregulated under similar condition. These genes are involved in chlorophyll synthesis, osmostress, oxidative, biotic stress, multi-stress tolerance including pathogen attack, and in mRNA degradation triggered by genotoxic stress [59][60][61][62][63][64][65] . We propose that these seven genes are acting as defence genes become mildly upregulated in case of rice plant colonized by KD-PiHOG1 P. indica as a result less colonization was found which leads to a loss of the benefits for the colonized plant or even to less growth and biomass production comparative to WT P. indica colonized rice plants.
In addition, PiHOG1 also gets phosphorylated upon stress exposure during colonized stage. We observed that HOG1 phosphorylation was found to be delayed and decreased in case of KD-PiHOG1 P. indica as compared to the WT P. indica colonizing host plant under salinity stress. The decreased phosphorylation event might be due to PiHOG1 down-regulation in KD-PiHOG1 P. indica, also upstream MAP kinase genes such as PiPBS2 and PiSSK2 were found to be down-regulated during colonization of KD-PiHOG1 P. indica with the host plant which suggests that PiHOG1 is involved in signalling related to the salinity tolerance and osmoregulation capacity of P. indica (Fig. 6C). Our findings provide the first evidence for the response of the beneficial root endophyte P. indica during osmostress as well as the role of the PiHOG1 in providing help to the colonized plant to overcome salinity stress. Thus, we propose that P. indica PiHOG1 could be a novel candidate to improve crop production in saline soil.
Identification, isolation and cloning of PiHOG1. P. indica HOG1 gene sequence was retrieved from P. indica genome (http://www.ncbi.nlm.nih.gov) by using tBLASTn and S. cerevisiae HOG1 protein amino acid sequence from the Saccharomycete Genome Database (SGD, http://www.yeastgenome.org/) as a query. Primers were designed using retrieved putative PiHOG1 sequence as template. PiHOG1 was PCR amplified using following program: 95 °C 2 min, 35 cycles (95 °C 1 min − 56 °C 30 sec − 72 °C 1 min 30 sec), 72 °C 5 min. PiHOG1 was cloned into pGEMT-easy vector (Promega, Finland). Cloning of the desired gene was confirmed by EcoRI restriction enzyme digestion and sequencing. Primers used for cloning and for the identification PiHOG1 genomic region and PiHOG1 cDNA are listed in Table S3.
Isolation of RNA and cDNA synthesis. P. indica was grown in 100 ml of KF medium for 5-7 days.
Fungal mycelium was filtered and transferred in to a fresh tube containing MN medium with 0.5 M NaCl and was incubated for 60 min. For RNA isolation, 0.2 g fungal tissue was crushed in liquid nitrogen and extracted with TRIZOL reagent 6 . RNA was treated with DNase I (Fermentas), incubated at 37 °C for 30 min and DNase I Scientific RepoRts | 6:36765 | DOI: 10.1038/srep36765 inactivation was done at 65 °C for 10 min. The cDNA synthesis was performed with Reverse Transcriptase (200 U, Fermentas) according to the manufacturer's instruction. This cDNA was used for the q-RT-PCR.
Phylogenetic tree of P. indica PiHOG1 was constructed by the neighbour-joining (N-J) method using the MEGA7 software (http://www.megasoftware.net/) 67 . Two types of phylogenetic analyses were constructed, one with homologs proteins of HOG1 from kingdom Fungi only (Table S4) and second with stress-activated MAP kinases (SAMAPKs), HOG1, Ser-Thr Kinases (STKs), Stress-induced MAP kinases (SIMPKs), P38 and Sty1 proteins from closely related as well as different groups like fungi, mammals, insects and plants (Table S5).
Complementation assay and spot test. For this purpose, heterologous system yeast ∆hog1 mutant strain was used (Euroscarf accession no. Y02724). The PiHOG1 cDNA insert was cloned into the BamHI and XhoI sites of the yeast expression vector pRS426GPD (Fig. S10) by using sequence specific primers (Table S3). The ∆ hog1 mutant was transformed with the recombinant pRS426GPD vector 66 after purification of the PiHOG1 insert by the LiCl-acetate method 68,69 . ∆ hog1 mutant cells transformed with empty vector was used as a control. S. cerevisiae WT strain was grown in 5 ml YPD medium while the ∆ hog1 + pRS426GPD and the ∆ hog1 + pRS426GPD-PiHOG1 mutant strain were grown overnight in SD-URA − liquid medium at 28 °C. For the complementation experiment, 2% YPD agar plates were prepared for of the different salinity conditions to spot serially diluted cells. Standard YPD medium was used as control. Four yeast strains used were as follow: wild type (wt), mutant yeast strain (∆ hog1), mutant yeast strain carrying the empty pRS426GPD plasmid (∆hog1 + pRS426GPD) and mutant yeast strain carrying the pRS426GPD plasmid with PiHOG1 gene under the GPD promoter (∆hog1 + pRS426GPD-PiHOG1). Freshly streaked cells were suspended in normal saline (0.9% NaCl) to an optical density at 600 nm (OD 600 ) of 0.1 (corresponds to approx. 1 × 10 6 cells/ml of yeast cells) and 10-fold serial dilutions were made in 0.9% saline. Intercellular glycerol content measurement. Yeast cells (WT BY4741, ∆hog1, ∆hog1 + pRS426GPD, ∆hog1 + pRS426GPD-PiHOG1) were cultured at 30 °C in SD medium and harvested at the early exponential phase (OD 600 = 0.5-0.8). Subsequently, cells were resuspended in new media with or without 0.5 M NaCl. After incubation for 1 h at 30 °C, cells were harvested and prepared as described 70 . Glycerol content was determined according to the application manual of the EnzyChrom ™ Glycerol Assay Kit (BioAssay Systems, USA) and by using microplate reader (SPECTRAmax M2 ROM v2.00c73).
Role of PiHOG1 in salinity tolerance capacity of rice plant during colonization. Development of KD-PiHOG1 P. indica (RNAi cassette formation, transformation, selection of transformants, q-RT-PCR and Northern blot). For this purpose, a ~350-bp unique fragment of PiHOG1 was selected using the BLAST tool. This unique fragment was amplified using the specific primers (Table S1). This PCR amplified 350-bp insert was subcloned into pRNAi vector at the unique EcoRV site (Fig. S11). This construct was named pRNAi-PiHOG1. Empty pRNAi and pRNAi-PiHOG1 was introduced into the P. indica mycelium by using electroporation 6 . Transformants were selected on Hygromycin which was used as a selection marker. Out of four colonies we have selected three transformants viz., TC1, TC2 and TC3 (Fig. S12Ai). The success of transformation was also confirmed by PCR using Hygromycin gene specific primer (Table S1). In all three selected transformants a band of approx. 600 bp was observed and no band was observed in case of WT P. indica (Fig. S12Aii). All three were tested for the expression of PiHOG1 by q-RT-PCR. We found that PiHOG1 transcripts level was reduced in all three transformed colonies. However, the least expression of PiHOG1 was found in case of TC3 (Fig. S12B). After checking the PiHog1 transcripts abundance analysis in selected colony, Northern blot was performed for siRNA analysis to check whether KD construct leads to siRNA accumulation or not. To do this, small RNAs were extracted and probed as described 6 . For this purpose, total RNA was isolated from KD-PiHOG1 P. indica (TC3 in duplicate) by using TRIzol reagent. Probe was prepared by end labeling of the PiHOG1 primer (5′ gagatgcttgagggcaaacc) using [γ -32P ]ATP and polynucleotide kinase as per the instructions described in manual (Molecular Labeling and Detection, Fermentas). The hybridization and autoradiography was performed as described previously 6 . Accumulation of siRNA was observed in the Northern blot in the case of KD-PiHOG1 P. indica (Fig. S12C). As TC3 showing lowest PiHOG1 transcript and SiRNA accumulation, it was further used for osmotolerance, colonization and expression analysis of HOG pathway related and salinity tolerance genes of P. indica and plant during interaction.
Furthermore, TC3 showed highest silencing of PiHOG1expression upon osmostress shock of 1 hr compared to WT P. indica (Fig. S13). Growth of TC3 colony was also analyzed in KF broth and on KF agar plates. We found that both WT and TC3 colony grow in similar fashion on KF media without Hygromycin, however no growth of WT P. indica was observed as compared to TC3 when grown in KF supplemented with Hygromycin (Fig. S14).
Comparative radial growth of WT P. indica and KD-PiHOG1 P. indica transformant was measured. It was found that both P. indica strains were growing normally on normal KF plates {Fig. S4A(a,b)} whereas under osmostress conditions i.e. 100 mM NaCl, 100 mM KCl, 100 mM MgCl 2 , 100 mM CaCl 2 and 300 mM Sorbitol, both WT and TC3, showed retarded growth {Fig. S4A(c-l)}. Growth of TC3 was reduced more as compared to WT P. indica (Fig. S4B). This selected TC3 was named as "KD-PiHOG1" and was used for further experiments.
Scientific RepoRts | 6:36765 | DOI: 10.1038/srep36765 Plant growth conditions, P. indica colonization and salinity treatment. The salinity sensitive rice variety Oryza sativa L. IR64 seeds were surface-sterilized and germinated on water-agar plates (0.8% Bacto Agar, Difco) at 25 °C in the dark for 3 days. Seven to ten seedlings were placed in pots (9 cm height by 10 cm diameter) containing sand (2-4 mm diameter). Plants were weekly supplied with half-strength modified Hoagland solution 6 . Three days-old germinated seedlings were planted in pots without P. indica and allowed to grow for 7 days. Rice seedlings were taken out and the roots were washed and were inoculated with the mycelium of WT P. indica and P. indica transformed with KD-PiHOG1 with sterile sand mixed with the fungal mycelium (1% in sand by w/w). Control plants were mock inoculated with autoclaved dH 2 O-mixed sand. P. indica colonization was checked 15 dpi under the light microscope (Leica type 020-518.500) 71 . In brief, colonization was checked by taking 10 root samples randomly from 3 different inoculated rice plants 15 dpi, i.e. when the rice seedlings were 25 days old. Initially, the rice root samples were softened in 10% KOH solution for 15 min, acidified with 1 M HCl for 10 min, and finally stained with 0.02% Trypan blue overnight. The samples were distained with 50% lacto-phenol for 1-2 h prior to observation under the light microscope. Distribution of intracellular chlamydospores within the cortex region of root was taken as a symptom of colonization. The percentage colonization of the full root length was calculated for the inoculated plants as per the following formula; Percent colonization = (Number of colonized root segments/Total number of segments) × 100 71,72 .
In order to check the role of PiHOG1 in P. indica associated stress tolerance during salinity stress and colonization with host plant, initially, 10 days old plants were given salinity treatment and this was considered as day 0. Pots having 7 plants were placed in trays with 200 mM salt solution. Following six sets were prepared viz., (1) Non-colonized plants without salinity treatment (2) 73 .
Proline content was measured at dpi 0 and 15. Plants were 10 and 25 days old at these measuring points. In brief, 0.5 g of plant material was homogenized in 10 ml of 3% aqueous sulfosalicylic acid and the mixture was centrifuged (10000 rpm, 10 min). Supernatant obtained was boiled with 2 ml acid ninhydrin and 2 ml of glacial acetic acid in a tightly closed glass tube for 1 h at 100 °C and the reaction was terminated using in ice. This mixture was extracted with 4 ml ice cold toluene with vigorous for 15-20 sec. The chromophore containing toluene was finally separated from the aqueous phase, warmed to room temperature and the absorbance was determined at 520 nm using toluene as blank. Proline concentration was determined from a standard curve and calculated on a fresh weight basis as follows: [(μ g proline/ml*ml toluene)/115.5 μ g/μ mole]/(g sample/5) = μ mole proline/g of fresh weight material. Proline content was measured according to the method described previously 74 . Expression analysis of salinity tolerance, HOG pathway genes of P. indica and salinity tolerance genes of rice plant (Quantitative RT-PCR). To find out the expression of genes of P. indica and rice plants during colonization, RNA was isolated from the non-colonized and colonized P. indica and rice plants. For this purpose, P. indica mycelia were grown in KF media for 7 days, filtered in minimal media and further grown for 3 days. Salinity treatment was given to acclimatized fungus by adding 0.5 M NaCl in MN medium (0.4 mM NaCl, 2.0 mM KH 2 PO 4 , 0.3 mM (NH 4 ) 2 HPO 4 , 0.6 mM CaCl 2 , 0.6 mM MgSO 4 , 3.6 mM FeCl 3 , 0.2 mM Thiaminehydrochloride, 0.1% (w/v) Trypticase peptone, 1% (w/v) Glucose, 5% (w/v) Malt extract, 2 mM KCl, 1 mM H 3 BO 3 , 0.22 mM MnSO 4 .H 2 O, 0.08 mM ZnSO 4 , 0.021 mM CuSO 4 , pH 5.8). After 1 hr of salinity stress treatment, fungus was immediately filtered and stored in liquid nitrogen. In case of colonization, plant roots were submerged in MN media supplemented with 0.5 M NaCl for 1 hr and the samples were frozen immediately and the total RNA was isolated.
HOG pathway related (PiHOG1, PiPBS2 and PiSSK2), regulated (PiGPD, PiSTL1, PiPFK26) and other osmoresponsive genes (PiHSP78, PiGRE2, PiENA1, PiPMR1 and PiPMC1) were explored by BLASTp search in the P. indica genome browser using the S. cerevisiae genes from the Saccharomyces Genome Database (SGD, http://www.yeastgenome.org/) as query. P. indica salinity tolerance genes 22 and rice salinity tolerance genes 67,68 were also selected for this study. The following cycles were used in the ABI 7500 Fast system (96 wells plates): pre-incubation at 95 °C for 5 min, denaturation 94 °C for 10 sec (4.8 C/s), annealing at 60 °C for 10 sec (2.5 °C/s), extension at 72 °C for 10 sec (4.8 °C/s), 40 cycles of amplification and final extension at 72 °C for 3 min. The Ct values were automatically calculated, the transcript levels were normalized against PiTef expression in case of P. indica 74 and against OsGAPDH in case of rice and the fold change was calculated based on the non-treated control. Two-step Real time-PCR protocol was used in different conditions. Real time-PCR reactions were performed in an ABI 7500 Fast sequence detection system (Applied Biosystems, Life Technologies, USA). The Fold Change values were calculated using the expression, where ∆ ∆ C T represents ∆ C T condition of interest gene-∆ C T control gene. The fold expression was calculated according to the 2 −∆∆C T method mentioned elsewhere 75 . The primers used in this study are shown in Table S3.
Scientific RepoRts | 6:36765 | DOI: 10.1038/srep36765 Phosphorylation detection during P. indica and plant interaction. P. indica was grown in KF broth media for 5 days at 30 ± 2 °C temperature and 110 rpm. In case of colonized plants, 25 dpi plants were taken. 0.5 M NaCl was added to the fungal culture or colonized roots and further incubated for 15, 30 and 60 min. The fungal mycelia or roots were quickly collected different time points of post salt addition and frozen in liquid nitrogen. For protein isolation 0.5 g fungal mycelia or roots were homogenised and extracted with 300 ml lysis buffer [(50 mM Tris-HCL (pH-7.5), 100 mM NaCl, 1% Triton X-100, 1 mM DTT, 10% glycerol)+ protease inhibitor cocktail (Calbiochem, Millipore, Germany) and phosphatase inhibitor cocktail (Biobasic, Canada) was added and the mixture was vortexed and centrifuged (12000 rcf, 15 min, 4 °C). Supernatant was collected and stored at − 80 °C. For Western blot analysis of PiHOG1 protein phosphorylation, protein content was separated by SDS-polyacrylamide gel. Ten μ g of protein was loaded on a 10% SDS-PAGE. Proteins were electrophoretically transferred to PVDF membrane by using Mini Trans-Blot ® Electrophoretic Transfer Cell (Bio-Rad). Blot was probed with 1:5000 dilutions of polyclonal Anti-phospho-p38 MAPK (pThr180/Tyr182; Signalway Antibody, USA) for 16 hours at 4 °C. After 3 washings, blot was probed with secondary Goat Anti-Rabbit IgG antibody (1:10000 dilutions) conjugated with horseradish peroxidase (HRP). Blot was developed with Clarity TM Western ECL substrate kit (BioRad) using Hyper processor TM (Amersham).