The OXI1 kinase pathway mediates Piriformospora indica-induced growth promotion in Arabidopsis.

Piriformospora indica is an endophytic fungus that colonizes roots of many plant species and promotes growth and resistance to certain plant pathogens. Despite its potential use in agriculture, little is known on the molecular basis of this beneficial plant-fungal interaction. In a genetic screen for plants, which do not show a P. indica- induced growth response, we isolated an Arabidopsis mutant in the OXI1 (Oxidative Signal Inducible1) gene. OXI1 has been characterized as a protein kinase which plays a role in pathogen response and is regulated by H2O2 and PDK1 (3-PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE1). A genetic analysis showed that double mutants of the two closely related PDK1.1 and PDK1.2 genes are defective in the growth response to P. indica. While OXI1 and PDK1 gene expression is upregulated in P. indica-colonized roots, defense genes are downregulated, indicating that the fungus suppresses plant defense reactions. PDK1 is activated by phosphatidic acid (PA) and P. indica triggers PA synthesis in Arabidopsis plants. Under beneficial co-cultivation conditions, H2O2 formation is even reduced by the fungus. Importantly, phospholipase D (PLD)α1 or PLDδ mutants, which are impaired in PA synthesis do not show growth promotion in response to fungal infection. These data establish that the P. indica-stimulated growth response is mediated by a pathway consisting of the PLD-PDK1-OXI1 cascade.

Arabidopsis MAP Kinase Phosphatase 1 (AtMKP1) negatively regulates MPK6-mediated PAMP responses and resistance against bacteria.

A primary component of plant defense is the detection of pathogen-associated molecular patterns (PAMPs) by plasma membrane-localized pathogen recognition receptors. PAMP perception results in rapid and transient activation of phosphorylation-dependent signaling pathways that lead to a wide array of defense-related responses, including extensive changes in gene expression. In Arabidopsis, several kinases, including the mitogen-activated protein kinases (MAPKs) MPK6 and MPK3, are rapidly activated after PAMP treatment, and are thought to positively regulate a wide array of defense-related responses. In contrast, negative regulation of PAMP responses by downstream phosphatases remains poorly understood. Here we report the identification of Arabidopsis MAP Kinase Phosphatase 1 (MKP1) as a negative regulator of diverse PAMP responses, including activation of MPK6 and MPK3, transient production of extracellular reactive oxygen species, accumulation of a subset of PAMP-regulated transcripts, and inhibition of seedling growth. In agreement with the enhanced PAMP response phenotypes observed in the mkp1 mutant, we found that mkp1 seedlings and adult plants are more resistant to the virulent bacterial pathogen Pseudomonas syringae pv. tomato (Pto) DC3000. Further genetic analysis revealed that MPK6, but not MPK3, is required for the mkp1-dependent increase in resistance to Pto and enhanced PAMP-induced growth inhibition observed in mkp1 seedlings. Together, our data support a role for MKP1 as a negative regulator of MPK6-mediated PAMP responses.

The role of auxins and cytokinins in the mutualistic interaction between Arabidopsis and Piriformospora indica.

Arabidopsis growth and reproduction are stimulated by the endophytic fungus Piriformospora indica. The fungus produces low amounts of auxins, but the auxin levels and the expression of auxin-regulated genes are not altered in colonized roots. Also, mutants with reduced auxin levels (ilr1-1, nit1-3, tfl2, cyp79 b2b3) respond to P. indica. However, the fungus rescues the dwarf phenotype of the auxin overproducer sur1-1 by converting free auxin into conjugates, which also results in the downregulation of the auxin-induced IAA6 and the upregulation of the P. indica-induced LRR1 gene. The fungus produces relatively high levels of cytokinins, and the cytokinin levels are higher in colonized roots compared with the uncolonized controls. trans-Zeatin cytokinin biosynthesis and the CRE1/AHK2 receptor combination are crucial for P. indica-mediated growth stimulation, while mutants lacking cis-zeatin, impaired in other cytokinin receptor combinations, or containing reduced cytokinin levels respond to the fungus. Since root colonization is not affected in the cytokinin mutants, we propose that cytokinins are required for P. indica-induced growth promotion. Finally, a comparative analysis of the phytohormone mutants allows the conclusion that the response to P. indica is independent of the architecture and size of the roots.

A leucine-rich repeat protein is required for growth promotion and enhanced seed production mediated by the endophytic fungus Piriformospora indica in Arabidopsis thaliana.

Piriformospora indica, a basidiomycete of the Sebacinaceae family, promotes the growth, development and seed production of a variety of plant species. Arabidopsis plants colonized with the fungus produce 22% more seeds than uncolonized plants. Deactivating the Arabidopsis single-copy gene DMI-1, which encodes an ion carrier required for mycorrihiza formation in legumes, does not affect the beneficial interaction between the two symbiotic partners. We used cellular and molecular responses initiated during the establishment of the interaction between P. indica and Arabidopsis roots to isolate mutants that fail to respond to the fungus. An ethylmethane sulfonate mutant (Piriformospora indica-insensitive-2; pii-2), and a corresponding insertion line, are impaired in a leucine-rich repeat protein (At1g13230). The protein pii-2, which contains a putative endoplasmic reticulum retention signal, is also found in Triton X-100-insoluble plasma membrane microdomains, suggesting that it is present in the endoplasmic reticulum/plasma membrane continuum in Arabidopsis roots. The microdomains also contain an atypical receptor protein (At5g16590) containing leucine-rich repeats, the message of which is transiently upregulated in Arabidopsis roots in response to P. indica. This response is not detectable in At1g13230 mutants, and the protein is not detectable in the At1g13230 mutant microdomains. Partial deactivation of a gene for a sphingosine kinase, which is required for the biosynthesis of sphingolipid found in plasma membrane microdomains, also affects the Arabidopsis/P. indica interaction. Thus, pii-2, and presumably also At5g16590, two proteins present in plasma membrane microdomains, appear to be involved in P. indica-induced growth promotion and enhanced seed production in Arabidopsis thaliana.

Expression of a receptor kinase in Arabidopsis roots is stimulated by the basidiomycete Piriformospora indica and the protein accumulates in Triton X-100 insoluble plasma membrane microdomains.

Piriformospora indica, an endophytic fungus of the Sebacinaceae family, colonises the roots of a wide variety of plant species and promotes their growth, in a manner similar to mycorrhizal fungi. We demonstrate that the fungus also interacts with the non-mycorrhizal host Arabidopsis thaliana. Promotion of root growth was detectable even before noticeable root colonization, and was accompanied by a massive transfer of phosphate from the media to the aerial parts of the seedlings. During the recognition period of both organisms, the message for a receptor kinase with leucine-rich repeats is transiently upregulated. The kinase is located in Triton X-100-insoluble plasma membrane microdomains. Thus, this is one of the earliest events of a plant root in response to a fungus reported to date.

The endophytic fungus Piriformospora indica stimulates the expression of nitrate reductase and the starch-degrading enzyme glucan-water dikinase in tobacco and Arabidopsis roots through a homeodomain transcription factor that binds to a conserved motif in their promoters.

Piriformospora indica, an endophytic fungus of the Sebacinaceae family, promotes growth of Arabidopsis and tobacco seedlings and stimulates nitrogen accumulation and the expression of the genes for nitrate reductase and the starch-degrading enzyme glucan-water dikinase (SEX1) in roots. Neither growth promotion nor stimulation of the two enzymes requires heterotrimeric G proteins. P. indica also stimulates the expression of the uidA gene under the control of the Arabidopsis nitrate reductase (Nia2) promoter in transgenic tobacco seedlings. At least two regions (-470/-439 and -103/-89) are important for Nia2 promoter activity in tobacco roots. One of the regions contains an element, ATGATAGATAAT, that binds to a homeodomain transcription factor in vitro. The message for this transcription factor is up-regulated by P. indica. The transcription factor also binds to a CTGATAGATCT segment in the SEX1 promoter in vitro. We propose that the growth-promoting effect initiated by P. indica is accompanied by a co-regulated stimulation of enzymes involved in nitrate and starch metabolisms.

Cytokinin stimulates polyribosome loading of nuclear-encoded mRNAs for the plastid ATP synthase in etioplasts of Lupinus luteus: the complex accumulates in the inner-envelope membrane with the CF(1) moiety located towards the stromal space.

Three of the nine subunits of the plastid ATP synthase, including the subunit of the CF(1) moiety (gene AtpC), are encoded in the nucleus. Application of cytokinin to etiolated lupine seedlings induces polyribosome association of their mRNAs. This appears to be specific as no such regulation was observed for messages for three ribosomal proteins. Cytokinin-mediated polyribosome loading was also observed for the spinach AtpC message in etiolated transgenic tobacco seedlings. Analysis of various spinach AtpC mRNA derivatives uncovered that the 5' untranslated region (5' UTR) of this message is sufficient to direct polyribosome loading, and that sequences at the 3' end of the AtpC 5' UTR, including an UC-rich motif, are crucial for this regulation. The increase in polyribosome loading of the AtpC message correlated with an increased synthesis of the polypeptide. The subunit, together with the ATP synthase complex, accumulates in the inner-envelope membrane with the CF(1) moiety located towards the stromal space of the etioplast. These results suggest that cytokinin promotes accumulation of the ATP synthase in the inner-envelope membrane of lupine etioplasts by stimulating the translation efficiency of their nuclear-encoded messages.