The mutualistic, endophytic fungus Piriformospora indica has been shown to confer biotic and abiotic stress tolerance to host plants. In this study, we investigated the impact of P. indica on the growth of Arabidopsis plants under normal and salt stress conditions. Our results demonstrate that P. indica colonization increases plant biomass, lateral roots density, and chlorophyll content under both conditions. Colonization with P. indica under salt stress was accompanied by a lower Na+/K+ ratio and less pronounced accumulation of anthocyanin, compared to control plants. Moreover, P. indica colonized roots under salt stress showed enhanced transcript levels of the genes encoding the high Affinity Potassium Transporter 1 (HKT1) and the inward-rectifying K+ channels KAT1 and KAT2, which play key roles in regulating Na+ and K+ homeostasis. The effect of P. indica colonization on AtHKT1;1 expression was also confirmed in the Arabidopsis line gl1-HKT:AtHKT1;1 that expresses an additional AtHKT1;1 copy driven by the native promoter. Colonization of the gl1-HKT:AtHKT1;1 by P. indica also increased lateral roots density and led to a better Na+/K+ ratio, which may be attributed to the observed increase in KAT1 and KAT2 transcript levels. Our findings demonstrate that P. indica colonization promotes Arabidopsis growth under salt stress conditions and that this effect is likely caused by modulation of the expression levels of the major Na+ and K+ ion channels, which allows establishing a balanced ion homeostasis of Na+/K+ under salt stress conditions.

Root Knot Nematode (RKN, Meloidogyne incognita) is one of the greatest damaging soil pathogens causes severe yield losses in cucumber and many other economic crops. Here, we evaluated the potential antagonistic effect of the root mutualistic fungus Piriformospora indica against RKN and their impact on vegetative growth, yield, photosynthesis, endogenous salicylic acid (SA) and its responsive genes. Our results showed that P. indica dramatically decreased the damage on shoot and root architecture of cucumber plants, which consequently enhanced yield of infested plants. Likewise, P. indica colonization clearly improved the chlorophyll content and delimited the negative impact of RNK on photosynthesis. Moreover, P. indica colonization exhibited a significant reduction of different vital nematological parameters such as soil larva density, amount of eggs/eggmass, eggmasses, females and amount of galls at cucumber roots. Additionally, the results showed that SA level was significantly increased generally in the roots of all treatments especially in plants infested with RKN alone as compared to control. This suggests that P. indica promoting SA levels in host cucumber plant roots to antagonize the RKN and alleviate severity damages occurred in its roots. This higher levels of SA in cucumber roots was consistent with the higher expressional levels of SA pathway genes PR1 and PR3. Furthermore, P. indica colonization reduces PR1, PR3 and increased NPR1 in roots of RKN infested cucumber plants when compared to non-colonized plants. Interestingly, our in vitro results showed that direct application of P. indica suspension against the J2s exhibited a significant increase in mortality ratio. Our results collectively suggest that P. indica promoting morphological, physiological and SA levels that might together play a major important role to alleviate the adverse impact of RKN in cucumber.

The root endophytic fungus Piriformospora indica plays an important role in increasing abiotic stress tolerance of its host plants. To explore the impact of P. indica on freezing tolerance, Arabidopsis seedlings were co-cultivated with P. indica exposed to −6°C for 6 h. Freezing stress decreased the survival rate, electrolyte leakage, leaf temperature, water potential and chlorophyll fluorescence of Arabidopsis plants in comparison to the controls. P. indica colonizion reduced the negative effects of freezing, and the plants contained also higher amounts of soluble proteins, proline and ascorbic acid during the post-thaw recovery period (4°C; 12 h). In contrast, the H₂O₂ and malondialdehyde levels were reduced in seedlings colonized by the fungus. The brassinolide (BR) and abscisic acid (ABA) levels dramatically increased and the transcript levels of several crucial freezing-stress related genes (CBFs, CORs, BZR1, SAG1 and PYL6) were higher in inoculated plants during the post-thaw recovery period. Finally, inocculated mutants impaired in the freezing tolerance response (such as ice1 for INDUCER OF CBF EXPRESSION1, a crucial basic helix-loop-helix transcription factor for the cold-response pathway in Arabidopsis, cbf1, −2, −3 for C-REPEAT-Binding Factor, cor47 and −15 for COLD-REGULATED and siz1 encoding the SUMO E3 LIGASE) showed better survival rates and higher expression levels of freezing-related target genes after freezing compared to the inocculated controls. Our results demonstrate that P. indica confers freezing tolerance and better post-thaw recovery in Arabidopsis, and stimulates the expression of several genes involved in the CBF-dependent pathway.

Water stress is the most critical aspect restricting the development of agriculture in regions with scarce water resources, which requires enhancing irrigation water-saving strategies. The current work discusses the potential application of the plant-strengthening root endophyte Piriformospora indica against moderate (25% less irrigation water) and severe (50% less irrigation water) water stress in comparison to the optimum irrigation conditions of greenhouse cucumbers. P. indica improved growth, nutrient content, and photosynthesis apparatus under normal or water-stress conditions. On the other hand, moderate and severe water stress reduced yield up to 47% and 83%, respectively, in non-colonized cucumber plants, while up to 28 and 78%, respectively, in P. indica-colonized plants. In terms of water-use efficiency (WUE), P. indica improved the WUE of colonized cucumber plants grown under moderate (26 L/kg) or severe stress (73 L/kg) by supporting colonized plants in producing higher yield per unit volume of water consumed by the crop in comparison to non-colonized plants under the same level of moderate (43 L/kg) or severe (81 L/kg) water stress. Furthermore, P. indica increased the indole-3-acetic acid (IAA) content, activity levels of catalase (CAT) and peroxidase (POD) with an apparent clear reduction in the abscisic acid (ABA), ethylene, malondialdehyde (MDA), proline contents and stomatal closure compared to non-stressed plants under both water-stress levels. In addition, chlorophyll a, b, a + b contents were increased in the leaves of the colonized plants under water-stress conditions. This improvement in chlorophyll content could be correlated with a significant increment in the transcripts of chlorophyll biosynthesis genes (protochlorophyllide oxidoreductase [POR], chlorophyll a oxygenase [CAO]) and a reduction in the chlorophyll degradation genes (PPH, pheophorbide a oxygenase [PAO], and red chlorophyll catabolite reductase [RCCR]). In conclusion, P. indica has the potential to enhance the cucumber yield grown under moderate water stress rather than severe water stress by improving WUE and altering the activity levels of antioxidant enzymes and chlorophyll metabolism-related genes.

Piriformospora indica is an endophytic fungus colonizing roots of a wide variety of plants. Previous studies showed that P. indica promotes early flowering and plant growth in the medicinal plant Coleus forskohlii. To determine the impact of P. indica on flowering time in Arabidopsis, we co-cultivated the plants with P. indica under long day condition. P. indica inoculated Arabidopsis plants displayed significant early flowering phenotype. qRT-PCR analysis of colonized plants revealed an up-regulation of flowering regulatory (FLOWERING LOCUS T, LEAFY, and APETALA1) and gibberellin biosynthetic (Gibberellin 20-Oxidase2, Gibberellin 3-Oxidase1 and Gibberellin requiring1) genes, while the flowering-repressing gene FLOWERING LOCUS C was down regulated. Quantification of gibberellins content showed that the colonization with P. indica caused an increase in GA4 content. Compared to wild-type plants, inoculation of the Arabidopsis ga5 mutant affected in gibberellin biosynthetic gene led to less pronounced changes in the expression of genes regulating flowering and to a lower increase in GA4 content. Taken together, our data indicate that P. indica promotes early flowering in Arabidopsis likely by increasing gibberellin content.