Sulfur transfer from the endophytic fungus Serendipita indica improves maize growth and requires the sulfate transporter SiSulT.

A deficiency of the essential macronutrient sulfur leads to stunted plant growth and yield loss; however, an association with a symbiotic fungus can greatly improve nutrient uptake by the host plant. Here, we identified and functionally characterized a high-affinity sulfate transporter from the endophytic fungus Serendipita indica. SiSulT fulfills all the criteria expected of a functional sulfate transporter responding to sulfur limitation: SiSulT expression was induced when S. indica was grown under low-sulfate conditions, and heterologous expression of SiSulT complemented a yeast mutant lacking sulfate transport. We generated a knockdown strain of SiSulT by RNA interference to investigate the consequences of the partial loss of this transporter for the fungus and the host plant (maize, Zea mays) during colonization. Wild-type (WT) S. indica, but not the knockdown strain (kd-SiSulT), largely compensated for low-sulfate availability and supported plant growth. Colonization by WT S. indica also allowed maize roots to allocate precious resources away from sulfate assimilation under low-sulfur conditions, as evidenced by the reduction in expression of most sulfate assimilation genes. Our study illustrates the utility of the endophyte S. indica in sulfur nutrition research and offers potential avenues for agronomically sound amelioration of plant growth in low-sulfate environments.

Piriformospora indica recruits host-derived putrescine for growth promotion in plants.

Growth promotion induced by the endosymbiont Piriformospora indica has been observed in various plants; however, except growth phytohormones, specific functional metabolites involved in P. indica-mediated growth promotion are unknown. Here, we used a gas chromatography-mass spectrometry-based untargeted metabolite analysis to identify tomato (Solanum lycopersicum) metabolites whose levels were altered during P. indica-mediated growth promotion. Metabolomic multivariate analysis revealed several primary metabolites with altered levels, with putrescine (Put) induced most significantly in roots during the interaction. Further, our results indicated that P. indica modulates the arginine decarboxylase (ADC)-mediated Put biosynthesis pathway via induction of SlADC1 in tomato. Piriformospora indica did not promote growth in Sladc1-(virus-induced gene silencing of SlADC1) lines of tomato and showed less colonization. Furthermore, using LC-MS/MS we showed that Put promoted growth by elevation of auxin (indole-3-acetic acid) and gibberellin (GA4 and GA7) levels in tomato. In Arabidopsis (Arabidopsis thaliana) adc knockout mutants, P. indica colonization also decreased and showed no plant growth promotion, and this response was rescued upon exogenous application of Put. Put is also important for hyphal growth of P. indica, indicating that it is co-adapted by both host and microbe. Taken together, we conclude that Put is an essential metabolite and its biosynthesis in plants is crucial for P. indica-mediated plant growth promotion and fungal growth.

Functional characterization of a high-affinity iron transporter (PiFTR) from the endophytic fungus Piriformospora indica and its role in plant growth and development.

Iron (Fe) is a micronutrient required for plant growth and development; however, most Fe forms in soil are not readily available to plants, resulting in low Fe contents in plants and, thereby, causing Fe deficiency in humans. Biofortification through plant-fungal co-cultivation might be a sustainable approach to increase crop Fe contents. Therefore, we aimed to examine the role of a Piriformospora indica Fe transporter on rice Fe uptake under low Fe conditions. A high-affinity Fe transporter (PiFTR) from P. indica was identified and functionally characterized. PiFTR fulfilled all criteria expected of a functional Fe transporter under Fe-limited conditions. Additionally, PiFTR expression was induced when P. indica was grown under low Fe conditions, and PiFTR complemented a yeast mutant lacking Fe transport. A knockdown (KD) P. indica strain was created via RNA interference to understand the physiological role of PiFTR. We observed that the KD-PiFTR-P. indica strain transported a significantly lower amount of Fe to colonized rice (Oryza sativa) than the wild type (WT) P. indica. WT P. indica-colonized rice plants were healthier and performed significantly better than KD-PiFTR-P. indica-colonized rice plants. Our study offers potential avenues for an agronomically sound amelioration of plant growth in low Fe environments.

Plant mineral transport systems and the potential for crop improvement.

MAIN CONCLUSION: Nutrient transporter genes could be a potential candidate for improving crop plants, with enhanced nutrient uptake leading to increased crop yield by providing tolerance against different biotic and abiotic stresses. The world's food supply is nearing a crisis in meeting the demands of an ever-growing global population, and an increase in both yield and nutrient value of major crops is vitally necessary to meet the increased population demand. Nutrients play an important role in plant metabolism as well as growth and development, and nutrient deficiency results in retarded plant growth and leads to reduced crop yield. A variety of cellular processes govern crop plant nutrient absorption from the soil. Among these, nutrient membrane transporters play an important role in the acquisition of nutrients from soil and transport of these nutrients to their target sites. In addition, as excess nutrient delivery has toxic effects on plant growth, these membrane transporters also play a significant role in the removal of excess nutrients in the crop plant. The key function provided by membrane transporters is the ability to supply the crop plant with an adequate level of tolerance against environmental stresses, such as soil acidity, alkalinity, salinity, drought, and pathogen attack. Membrane transporter genes have been utilized for the improvement of crop plants, with enhanced nutrient uptake leading to increased crop yield by providing tolerance against different biotic and abiotic stresses. Further understanding of the basic mechanisms of nutrient transport in crop plants could facilitate the advanced design of engineered plant crops to achieve increased yield and improve nutrient quality through the use of genetic technologies as well as molecular breeding. This review is focused on nutrient toxicity and tolerance mechanisms in crop plants to aid in understanding and addressing the anticipated global food demand.

Metal transporters in organelles and their roles in heavy metal transportation and sequestration mechanisms in plants.

Heavy metal toxicity is one of the major concerns for agriculture and health. Accumulation of toxic heavy metals at high concentrations in edible parts of crop plants is the primary cause of disease in humans and cattle. A dramatic increase in industrialization, urbanization, and other high anthropogenic activities has led to the accumulation of heavy metals in agricultural soil, which has consequently disrupted soil conditions and affected crop yield. By now, plants have developed several mechanisms to cope with heavy metal stress. However, not all plants are equally effective in dealing with the toxicity of high heavy metal concentrations. Plants have modified their anatomy, morphophysiology, and molecular networks to survive under changing environmental conditions. Heavy metal sequestration is one of the essential processes evolved by some plants to deal with heavy metals' toxic concentration. Some plants even have the ability to accumulate metals in high quantities in the shoots/organelles without toxic effects. For intercellular and interorganeller metal transport, plants harbor spatially distributed various transporters which mainly help in uptake, translocation, and redistribution of metals. This review discusses different heavy metal transporters in different organelles and their roles in metal sequestration and redistribution to help plants cope with heavy metal stress. A good understanding of the processes at stake helps in developing more tolerant crops without affecting their productivity.

Crosstalk between phytohormones and secondary metabolites in the drought stress tolerance of crop plants: A review.

Drought stress negatively affects crop performance and weakens global food security. It triggers the activation of downstream pathways, mainly through phytohormones homeostasis and their signaling networks, which further initiate the biosynthesis of secondary metabolites (SMs). Roots sense drought stress, the signal travels to the above-ground tissues to induce systemic phytohormones signaling. The systemic signals further trigger the biosynthesis of SMs and stomatal closure to prevent water loss. SMs primarily scavenge reactive oxygen species (ROS) to protect plants from lipid peroxidation and also perform additional defense-related functions. Moreover, drought-induced volatile SMs can alert the plant tissues to perform drought stress mitigating functions in plants. Other phytohormone-induced stress responses include cell wall and cuticle thickening, root and leaf morphology alteration, and anatomical changes of roots, stems, and leaves, which in turn minimize the oxidative stress, water loss, and other adverse effects of drought. Exogenous applications of phytohormones and genetic engineering of phytohormones signaling and biosynthesis pathways mitigate the drought stress effects. Direct modulation of the SMs biosynthetic pathway genes or indirect via phytohormones' regulation provides drought tolerance. Thus, phytohormones and SMs play key roles in plant development under the drought stress environment in crop plants.

Calcium channel CNGC19 mediates basal defense signaling to regulate colonization by Piriformospora indica in Arabidopsis roots.

The activation of calcium signaling is a crucial event for perceiving environmental stress. Colonization by Piriformospora indica, a growth-promoting root endosymbiont, activates cytosolic Ca2+ in Arabidopsis roots. In this study, we examined the role and functional relevance of calcium channels responsible for Ca2+ fluxes. Expression profiling revealed that CYCLIC NUCLEOTIDE GATED CHANNEL 19 (CNGC19) is an early-activated gene, induced by unidentified components in P. indica cell-wall extract. Functional analysis showed that loss-of-function of CNGC19 resulted in growth inhibition by P.indica, due to increased colonization and loss of controlled fungal growth. The cngc19 mutant showed reduced elevation of cytosolic Ca2+ in response to P. indica cell-wall extract in comparison to the wild-type. Microbe-associated molecular pattern-triggered immunity was compromised in the cngc19 lines, as evidenced by unaltered callose deposition, reduced cis-(+)-12-oxo-phytodienoic acid, jasmonate, and jasmonoyl isoleucine levels, and down-regulation of jasmonate and other defense-related genes, which contributed to a shift towards a pathogenic response. Loss-of-function of CNGC19 resulted in an inability to modulate indole glucosinolate content during P. indica colonization. CNGC19-mediated basal immunity was dependent on the AtPep receptor, PEPR. CNGC19 was also crucial for P. indica-mediated suppression of AtPep-induced immunity. Our results thus demonstrate that Arabidopsis CNGC19 is an important Ca2+ channel that maintains a robust innate immunity and is crucial for growth-promotion signaling upon colonization by P. indica.

Isolation of genes conferring salt tolerance from Piriformospora indica by random overexpression in Escherichia coli.

Piriformospora indica, a root endophytic fungus identified in the Indian Thar desert, colonizes the roots of plants and provides resistance towards biotic stress as well as tolerance to abiotic stress in the plants. Despite its positive impact on the host, little is known about the P. indica genes that are involved in salt stress tolerance. Therefore this study was conducted to identify and isolate high salinity-tolerance genes from P. indica. Thirty-six salinity-tolerance genes were obtained by functional screening, based on random over expression of a P. indica cDNA library in Escherichia coli grown on medium supplemented with 0.6 M NaCl. The salinity tolerance conferred by these 36 genes in bacteria was further confirmed by using another strain of E. coli (DH5α) transformants. However when the expression of these 36 genes was analysed in P. indica using quantitative RT-PCR, we found only six genes were up-regulated by salt stress. These six genes are involved in different cellular processes, such as metabolism, energy and biosynthetic processes, DNA repair, regulation of protein turnover, transport and salt stress tolerance. This work presents the basis for further molecular analyses of the mechanisms of salt tolerance in P. indica and for the use of this endophyte to confer salt tolerance to plants.

De novo genome sequencing and secretome analysis of Tilletia indica inciting Karnal bunt of wheat provides pathogenesis-related genes.

Tilletia indica is an internationally quarantined fungal pathogen causing Karnal bunt of wheat. The present study carried out that the whole genome of T. indica was sequenced and identified transposable elements, pathogenicity-related genes using a comparative genomics approach. The T. indica genome assembly size of 33.7 MB was generated using Illumina and Pac Bio platforms with GC content of 55.0%. A total of 1737 scaffolds were obtained with N50 of 58,667 bp. The ab initio gene prediction was performed using Ustilago maydis as the reference species. A total number of 10,113 genes were predicted with an average gene size of 1945 bp out of which functionally annotated genes were 7262. A total number of 3216 protein-coding genes were assigned in different categories. Out of a total number of 1877 transposable elements, gypsy had the highest count (573). Total 5772 simple sequence repeats were identified in the genome assembly, and the most abundant simple sequence repeat type was trinucleotide having 42% of total SSRs. The comparative genome analysis suggested 3751 proteins of T. indica had orthologs in five fungi, whereas 126 proteins were unique to T. indica. Secretome analysis revealed the presence of 1014 secretory proteins and few carbohydrate-active enzymes in the genome. Some putative candidate pathogenicity-related genes were identified in the genome. The whole genome of T. indica will provide a window to understand the pathogenesis mechanism, fungal life cycle, survival of teliospores, and novel strategies for management of Karnal bunt disease of wheat.

A Botrytis cinerea KLP-7 Kinesin acts as a Virulence Determinant during Plant Infection.

Botrytis cinerea is a necrotrophic pathogen that infects many important crops. In an attempt to unravel some novel factors that govern pathogenicity in B. cinerea, Agrobacterium tumefaciens mediated transformation (ATMT) was deployed, and a number of tagged transformants were generated. Among these, a mutant, BCM-29 exhibited slower growth rate, reduced conidia size, conidiation and penetration. The mutant was also defective in secretion of oxalic acid (OA) and exhibited reduced activities of polygalacturonase (PG) and pectin methyl esterases (PME). TAIL-PCR followed by BLAST search identified the tagged gene as KLP-7 that encodes for kinesin. Targeted deletion of KLP-7 resulted in several folds decrease in virulence of mutants as compared to WT, while complementation of the gene helped in rescue of virulence traits. This is the first time when a unique kinesin KLP-7 that is mainly found in the phylum Pezizomycotina has been linked to virulence in B. cinerea.

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.

Piriformospora indica rescues growth diminution of rice seedlings during high salt stress.

Piriformospora indica association has been reported to increase biotic as well as abiotic stress tolerance of its host plants. We analyzed the beneficial effect of P. indica association on rice seedlings during high salt stress conditions (200 and 300 mM NaCl). The growth parameters of rice seedlings such as root and shoot lengths or fresh and dry weights were found to be enhanced in P. indica-inoculated rice seedlings as compared with non-inoculated control seedlings, irrespective of whether they are exposed to salt stress or not. However, salt-stressed seedlings performed much better in the presence of the fungus compared with non-inoculated control seedlings. The photosynthetic pigment content [chlorophyll (Chl) a, Chl b, and carotenoids] was significantly higher in P. indica-inoculated rice seedlings under high salt stress conditions as compared with salt-treated non-inoculated rice seedlings, in which these pigments were found to be decreased. Proline accumulation was also observed during P. indica colonization, which may help the inoculated plants to become salt tolerant. Taken together, P. indica rescues growth diminution of rice seedlings under salt stress.