Sphingolipid-Induced Programmed Cell Death is a Salicylic Acid and EDS1-Dependent Phenotype in Arabidopsis Fatty Acid Hydroxylase (Fah1, Fah2) and Ceramide Synthase (Loh2) Triple Mutants.

Ceramides (Cers) and long-chain bases (LCBs) are plant sphingolipids involved in the induction of plant programmed cell death (PCD). The fatty acid hydroxylase mutant fah1 fah2 exhibits high Cer levels and moderately elevated LCB levels. Salicylic acid glucoside level is increased in this mutant, but no cell death can be detected by trypan blue staining. To determine the effect of Cers with different chain lengths, fah1 fah2 was crossed with ceramide synthase mutants longevity assurance gene one homologue1-3 (loh1, loh2 and loh3). Surprisingly, only triple mutants with loh2 show cell death detected by trypan blue staining under the selected conditions. Sphingolipid profiling revealed that the greatest differences between the triple mutant plants are in the LCB and LCB-phosphate (LCB-P) fraction. fah1 fah2 loh2 plants accumulate LCB d18:0, LCB t18:0 and LCB-P d18:0. Crossing fah1 fah2 loh2 with the salicylic acid (SA) synthesis mutant sid2-2 and with the SA signaling mutants enhanced disease susceptibility 1-2 (eds1-2) and phytoalexin deficient 4-1 (pad4-1) revealed that lesions are SA- and EDS1-dependent. These quadruple mutants also confirm that there may be a feedback loop between SA and sphingolipid metabolism as they accumulated less Cers and LCBs. In conclusion, PCD in fah1 fah2 loh2 is a SA- and EDS1-dependent phenotype, which is likely due to accumulation of LCBs.

Disruption of Arabidopsis neutral ceramidases 1 and 2 results in specific sphingolipid imbalances triggering different phytohormone-dependent plant cell death programmes.

Sphingolipids act as regulators of programmed cell death (PCD) and the plant defence response. The homeostasis between long-chain base (LCB) and ceramide (Cer) seems to play an important role in executions of PCD. Therefore, deciphering the role of neutral ceramidases (NCER) is crucial to identify the sphingolipid compounds that trigger and execute PCD. We performed comprehensive sphingolipid and phytohormone analyses of Arabidopsis ncer mutants, combined with gene expression profiling and microscopic analyses. While ncer1 exhibited early leaf senescence (developmentally controlled PCD - dPCD) and an increase in hydroxyceramides, ncer2 showed spontaneous cell death (pathogen-triggered PCD-like - pPCD) accompanied by an increase in LCB t18:0 at 35 d, respectively. Loss of NCER1 function resulted in accumulation of jasmonoyl-isoleucine (JA-Ile) in the leaves, whereas disruption of NCER2 was accompanied by higher levels of salicylic acid (SA) and increased sensitivity to Fumonisin B1 (FB1 ). All mutants were also found to activate plant defence pathways. These data strongly suggest that NCER1 hydrolyses ceramides whereas NCER2 functions as a ceramide synthase. Our results reveal an important role of NCER in the regulation of both dPCD and pPCD via a tight connection between the phytohormone and sphingolipid levels in these two processes.

Key Components of Different Plant Defense Pathways Are Dispensable for Powdery Mildew Resistance of the Arabidopsis mlo2 mlo6 mlo12 Triple Mutant.

Loss of function mutations of particular plant MILDEW RESISTANCE LOCUS O (MLO) genes confer durable and broad-spectrum penetration resistance against powdery mildew fungi. Here, we combined genetic, transcriptomic and metabolomic analyses to explore the defense mechanisms in the fully resistant Arabidopsis thaliana mlo2 mlo6 mlo12 triple mutant. We found that this genotype unexpectedly overcomes the requirement for indolic antimicrobials and defense-related secretion, which are critical for incomplete resistance of mlo2 single mutants. Comparative microarray-based transcriptome analysis of mlo2 mlo6 mlo12 mutants and wild type plants upon Golovinomyces orontii inoculation revealed an increased and accelerated accumulation of many defense-related transcripts. Despite the biotrophic nature of the interaction, this included the non-canonical activation of a jasmonic acid/ethylene-dependent transcriptional program. In contrast to a non-adapted powdery mildew pathogen, the adapted powdery mildew fungus is able to defeat the accumulation of defense-relevant indolic metabolites in a MLO protein-dependent manner. We suggest that a broad and fast activation of immune responses in mlo2 mlo6 mlo12 plants can compensate for the lack of single or few defense pathways. In addition, our results point to a role of Arabidopsis MLO2, MLO6, and MLO12 in enabling defense suppression during invasion by adapted powdery mildew fungi.

Optimized Jasmonic Acid Production by Lasiodiplodia theobromae Reveals Formation of Valuable Plant Secondary Metabolites.

Jasmonic acid is a plant hormone that can be produced by the fungus Lasiodiplodia theobromae via submerged fermentation. From a biotechnological perspective jasmonic acid is a valuable feedstock as its derivatives serve as important ingredients in different cosmetic products and in the future it may be used for pharmaceutical applications. The objective of this work was to improve the production of jasmonic acid by L. theobromae strain 2334. We observed that jasmonic acid formation is dependent on the culture volume. Moreover, cultures grown in medium containing potassium nitrate as nitrogen source produced higher amounts of jasmonic acid than analogous cultures supplemented with ammonium nitrate. When cultivated under optimal conditions for jasmonic acid production, L. theobromae secreted several secondary metabolites known from plants into the medium. Among those we found 3-oxo-2-(pent-2-enyl)-cyclopentane-1-butanoic acid (OPC-4) and hydroxy-jasmonic acid derivatives, respectively, suggesting that fungal jasmonate metabolism may involve similar reaction steps as that of plants. To characterize fungal growth and jasmonic acid-formation, we established a mathematical model describing both processes. This model may form the basis of industrial upscaling attempts. Importantly, it showed that jasmonic acid-formation is not associated to fungal growth. Therefore, this finding suggests that jasmonic acid, despite its enormous amount being produced upon fungal development, serves merely as secondary metabolite.

OPDA Has Key Role in Regulating Plant Susceptibility to the Root-Knot Nematode Meloidogyne hapla in Arabidopsis.

Jasmonic acid (JA) is a plant hormone that plays important roles in regulating plant defenses against necrotrophic pathogens and herbivorous insects, but the role of JA in mediating the plant responses to root-knot nematodes has been unclear. Here we show that an application of either methyl jasmonate (MeJA) or the JA-mimic coronatine (COR) on Arabidopsis significantly reduced the number of galls caused by the root-knot nematode Meloidogyne hapla. Interestingly, the MeJA-induced resistance was independent of the JA-receptor COI1 (CORONATINE INSENSITIVE 1). The MeJA-treated plants accumulated the JA precursor cis-(+)-12-oxo-phytodienoic acid (OPDA) in addition to JA/JA-Isoleucine, indicating a positive feedback loop in JA biosynthesis. Using mutants in the JA-biosynthetic pathway, we found that plants deficient in the biosynthesis of JA and OPDA were hyper-susceptible to M. hapla. However, the opr3 mutant, which cannot convert OPDA to JA, exhibited wild-type levels of nematode galling. In addition, mutants in the JA-biosynthesis and perception which lie downstream of opr3 also displayed wild-type levels of galling. The data put OPR3 (OPDA reductase 3) as the branch point between hyper-susceptibility and wild-type like levels of disease. Overall, the data suggests that the JA precursor, OPDA, plays a role in regulating plant defense against nematodes.

Analysis of plant-bacteria interactions in their native habitat: bacterial communities associated with wild tobacco are independent of endogenous jasmonic acid levels and developmental stages.

Jasmonic acid (JA) mediates defense responses against herbivores and necrotrophic pathogens but does it influence the recruitment of bacterial communities in the field? We conducted field and laboratory experiments with transformed Nicotiana attenuata plants deficient in jasmonate biosynthesis (irAOC) and empty vector controls (EV) to answer this question. Using both culture-dependent and independent techniques, we characterized root and leaf-associated bacterial communities over five developmental stages, from rosette through flowering of plants grown in their natural habitat. Based on the pyrosequencing results, alpha and beta diversity did not differ among EV and irAOC plants or over ontogeny, but some genera were more abundant in one of the genotypes. Furthermore, bacterial communities were significantly different among leaves and roots. Taxa isolated only from one or both plant genotypes and hence classified as 'specialists' and 'generalists' were used in laboratory tests to further evaluate the patterns observed from the field. The putative specialist taxa did not preferentially colonize the jasmonate-deficient genotype, or alter the plant's elicited phytohormone signaling. We conclude that in N. attenuata, JA signaling does not have a major effect on structuring the bacterial communities and infer that colonization of plant tissues is mainly shaped by the local soil community in which the plant grows.

Dimethyl disulfide produced by the naturally associated bacterium bacillus sp B55 promotes Nicotiana attenuata growth by enhancing sulfur nutrition.

Bacillus sp B55, a bacterium naturally associated with Nicotiana attenuata roots, promotes growth and survival of wild-type and, particularly, ethylene (ET)-insensitive (35)S-ethylene response1 (etr1) N. attenuata plants, which heterologously express the mutant Arabidopsis thaliana receptor ETR1-1. We found that the volatile organic compound (VOC) blend emitted by B55 promotes seedling growth, which is dominated by the S-containing compound dimethyl disulfide (DMDS). DMDS was depleted from the headspace during cocultivation with seedlings in bipartite Petri dishes, and (35)S was assimilated from the bacterial VOC bouquet and incorporated into plant proteins. In wild-type and (35)S-etr1 seedlings grown under different sulfate (SO(4)(-2)) supply conditions, exposure to synthetic DMDS led to genotype-dependent plant growth promotion effects. For the wild type, only S-starved seedlings benefited from DMDS exposure. By contrast, growth of (35)S-etr1 seedlings, which we demonstrate to have an unregulated S metabolism, increased at all SO(4)(-2) supply rates. Exposure to B55 VOCs and DMDS rescued many of the growth phenotypes exhibited by ET-insensitive plants, including the lack of root hairs, poor lateral root growth, and low chlorophyll content. DMDS supplementation significantly reduced the expression of S assimilation genes, as well as Met biosynthesis and recycling. We conclude that DMDS by B55 production is a plant growth promotion mechanism that likely enhances the availability of reduced S, which is particularly beneficial for wild-type plants growing in S-deficient soils and for (35)S-etr1 plants due to their impaired S uptake/assimilation/metabolism.

A native plant growth promoting bacterium, Bacillus sp. B55, rescues growth performance of an ethylene-insensitive plant genotype in nature.

Many plants have intimate relationships with soil microbes, which improve the plant's growth and fitness through a variety of mechanisms. Bacillus sp. isolates are natural root-associated bacteria, isolated from Nicotiana attenuata plant roots growing in native soils. A particular isolate B55, was found to have dramatic plant growth promotion (PGP) effects on wild type (WT) and transgenic plants impaired in ethylene (ET) perception (35S-etr1), the genotype from which this bacterium was first isolated. B55 not only improves N. attenuata growth under in vitro, glasshouse, and field conditions, but it also "rescues" many of the deleterious phenotypes associated with ET insensitivity. Most notably, B55 dramatically increases the growth and survival of 35S-etr1 plants under field conditions. To our knowledge, this is the first demonstration of a PGP effect in a native plant-microbe association under natural conditions. Our study demonstrates that this facultative mutualistic plant-microbe interaction should be viewed as part of the plant's extended phenotype. Possible modalities of recruitment and mechanisms of PGP are discussed.