Iron distribution through the developmental stages of Medicago truncatula nodules.

Paramount to symbiotic nitrogen fixation (SNF) is the synthesis of a number of metalloenzymes that use iron as a critical component of their catalytical core. Since this process is carried out by endosymbiotic rhizobia living in legume root nodules, the mechanisms involved in iron delivery to the rhizobia-containing cells are critical for SNF. In order to gain insight into iron transport to the nodule, we have used synchrotron-based X-ray fluorescence to determine the spatio-temporal distribution of this metal in nodules of the legume Medicago truncatula with hitherto unattained sensitivity and resolution. The data support a model in which iron is released from the vasculature into the apoplast of the infection/differentiation zone of the nodule (zone II). The infected cell subsequently takes up this apoplastic iron and delivers it to the symbiosome and the secretory system to synthesize ferroproteins. Upon senescence, iron is relocated to the vasculature to be reused by the shoot. These observations highlight the important role of yet to be discovered metal transporters in iron compartmentalization in the nodule and in the recovery of an essential and scarce nutrient for flowering and seed production

MtNramp1 mediates iron import in rhizobia-infected Medicago truncatula cells.

Symbiotic nitrogen fixation is a process that requires relatively high quantities of iron provided by the host legume. Using synchrotron-based X-ray fluorescence, we have determined that this iron is released from the vasculature into the apoplast of zone II of M. truncatula nodules. This overlaps with the distribution of MtNramp1, a plasma membrane iron importer. The importance of MtNramp1 in iron transport for nitrogen fixation is indicated by the 60% reduction of nitrogenase activity observed in knock-down lines, most likely due to deficient incorporation of this essential metal cofactor at the necessary levels

Insights of the <i>Neofusicoccum parvum</i>–<i>Liquidambar styraciflua</i> Interaction and Identification of New Cysteine-Rich Proteins in Both Species

Neofusicoccum parvum belongs to the Botryosphaeriaceae family, which contains endophytes and pathogens of woody plants. In this study, we isolated 11 strains from diseased tissue of Liquidambar styraciflua. Testing with Koch’s postulates—followed by a molecular approach—revealed that N. parvum was the most pathogenic strain. We established an in vitro pathosystem (L. styraciflua foliar tissue–N. parvum) in order to characterize the infection process during the first 16 days. New CysRPs were identified for both organisms using public transcriptomic and genomic databases, while mRNA expression of CysRPs was analyzed by RT-qPCR. The results showed that N. parvum caused disease symptoms after 24 h that intensified over time. Through in silico analysis, 5 CysRPs were identified for each organism, revealing that all of the proteins are potentially secreted and novel, including two of N. parvum proteins containing the CFEM domain. Interestingly, the levels of the CysRPs mRNAs change during the interaction. This study reports N. parvum as a pathogen of L. styraciflua for the first time and highlights the potential involvement of CysRPs in both organisms during this interaction

Medicago truncatula Zinc-Iron Permease6 provides zinc to rhizobia-infected nodule cells

International audienceZinc is a micronutrient required for symbiotic nitrogen fixation. It has been proposed that in model legume Medicago truncatula, zinc is delivered by the root vasculature into the nodule and released in the infection/differentiation zone. There, transporters must introduce this element into rhizobia-infected cells to metallate the apoproteins that use zinc as a cofactor. MtZIP6 (Medtr4g083570) is an M. truncatula Zinc-Iron Permease (ZIP) that is expressed only in roots and nodules, with the highest expression levels in the infection/differentiation zone. Immunolocalization studies indicate that it is located in the plasma membrane of nodule rhizobia-infected cells. Down-regulating MtZIP6 expression levels with RNAi does not result in any strong phenotype when plants are fed mineral nitrogen. However, these plants displayed severe growth defects when they depended on nitrogen fixed by their nodules, losing of 80% of their nitrogenase activity. The reduction of this activity was likely an indirect effect of zinc being retained in the infection/differentiation zone and not reaching the cytosol of rhizobia-infected cells. These data are consistent with a model in which MtZIP6 would be responsible for zinc uptake by rhizobia-infected nodule cells in the infection/differentiation zone

Molecular evidence of the avocado defense response to Fusarium kuroshium infection: a deep transcriptome analysis using RNA-Seq

Fusarium kuroshium is a novel member of the Ambrosia Fusarium Clade (AFC) that has been recognized as one of the symbionts of the invasive Kuroshio shot hole borer, an Asian ambrosia beetle. This complex is considered the causal agent of Fusarium dieback, a disease that has severely threatened natural forests, landscape trees, and avocado orchards in the last 8 years. Despite the interest in this species, the molecular responses of both the host and F. kuroshium during the infection process and disease establishment remain unknown. In this work, we established an in vitro pathosystem using Hass avocado stems inoculated with F. kuroshium to investigate differential gene expression at 1, 4, 7 and 14 days post-inoculation. RNA-seq technology allowed us to obtain data from both the plant and the fungus, and the sequences obtained from both organisms were analyzed independently. The pathosystem established was able to mimic Fusarium dieback symptoms, such as carbohydrate exudation, necrosis, and vascular tissue discoloration. The results provide interesting evidence regarding the genes that may play roles in the avocado defense response to Fusarium dieback disease. The avocado data set comprised a coding sequence collection of 51,379 UniGenes, from which 2,403 (4.67%) were identified as differentially expressed. The global expression analysis showed that F. kuroshium responsive UniGenes can be clustered into six groups according to their expression profiles. The biologically relevant functional categories that were identified included photosynthesis as well as responses to stress, hormones, abscisic acid, and water deprivation. Additionally, processes such as oxidation-reduction, organization and biogenesis of the cell wall and polysaccharide metabolism were detected. Moreover, we identified orthologues of nucleotide-binding leucine-rich receptors, and their possible action mode was analyzed. In F. kuroshium, we identified 57 differentially expressed genes. Interestingly, the alcohol metabolic process biological category had the highest number of upregulated genes, and the enzyme group in this category may play an important role in the mechanisms of secondary metabolite detoxification. Hydrolytic enzymes, such as endoglucanases and a pectate lyase, were also identified, as well as some proteases. In conclusion, our research was conducted mainly to explain how the vascular tissue of a recognized host of the ambrosia complex responds during F. kuroshium infection since Fusarium dieback is an ambrosia beetle-vectored disease and many variables facilitate its establishment

Plant growth-promoting rhizobacteria associated with avocado display antagonistic activity against <i>Phytophthora cinnamomi</i> through volatile emissions

<div><p>Rhizobacteria associated with crops constitute an important source of potentially beneficial microorganisms with plant growth promoting activity or antagonistic effects against phytopathogens. In this study, we evaluated the plant growth promoting activity of 11 bacterial isolates that were obtained from the rhizosphere of healthy avocado trees and from that of avocado trees having survived root rot infestations. Seven bacterial isolates, belonging to the genera <i>Bacillus</i>, <i>Pseudomonas</i> and <i>Arthrobacter</i>, promoted <i>in vitro</i> growth of <i>Arabidopsis thaliana</i>. These isolates were then tested for antagonistic activity against <i>Phytophthora cinnamomi</i>, in direct dual culture assays. Two of those rhizobacterial isolates, obtained from symptomatic-declining trees, displayed antagonistic activity. Isolate A8a, which is closely related to <i>Bacillus acidiceler</i>, was also able to inhibit <i>P</i>. <i>cinnamomi</i> growth <i>in vitro</i> by 76% through the production of volatile compounds. Solid phase microextraction (SPME) and analysis by gas chromatography coupled with mass spectrometry (GC-MS) allowed to tentatively identify the main volatiles emitted by isolate A8a as 2,3,5-trimethylpyrazine, 6,10-dimethyl-5,9-undecadien-2-one and 3-amino-1,3-oxazolidin-2-one. These volatile compounds have been reported to show antifungal activity when produced by other bacterial isolates. These results confirm the significance of rhizobacteria and suggest that these bacteria could be used for biocontrol of soil borne oomycetes through their volatiles emissions.</p></div

Co-cultivation of <i>Arabidopsis thaliana</i> seedlings with rhizobacterial isolates from symptomatic-declining avocado trees.

<p>Representative photographs of Arabidopsis Col-0 seedlings transferred to non-inoculated (Control) fresh media, or inoculated with rhizobacterial isolates at 2.5 cm (long distance) and 1 cm (close distance) from root tip. Primary root length (b), lateral root number (c) and fresh weight accumulation (d) were the developmental parameters analyzed. Data values represent the mean of 30 seedlings ± SE per treatment; different letters in graphs indicate significant differences (<i>P</i> < 0.05).</p

Antagonical activity of isolate A8a, phylogenetically close to <i>Bacillus acidiceler</i>, against <i>Phytophthora cinnamomi</i>.

<p>Disks of <i>P</i>. <i>cinnamomi</i> mycelium were grown on agar PDA-containing plates. Radial growth was monitored for 11 days in non-inoculated conditions (a). Confrontation with <i>B</i>. <i>acidiceler</i> was performed by directly co-cultivating mycelial disks with bacterial inoculum, at a distance of 2 cm (b), or indirectly, growing them on the opposite halves of the same Petri dish (c). Assays were carried out by triplicate. Representative plates were photographed at day 5 and 11 after inoculation (dai). At 7 dai, the inhibition percentage of mycelial radial growth by direct (light gray bars) or indirect (dark gray bars) antagonism was analyzed (d); hyphal deformations induced by indirect inoculation were visually analyzed by stereoscopic observations at 7 dai (e). Values shown in (d) represent the mean of three replicates ± SD; asterisks indicate significant inhibition (<i>P</i> ≤ 0.05).</p

Maximum-Likelihood tree of partially sequenced 16S rRNA genes.

<p>Bold letters indicate rhizobacterial isolates that were obtained in this study. Values above nodes are bootstrap values obtained from 1000 replicates.</p

Localization and characteristics of sampling site.

<p>Geographic projection of Veracruz state and the Municipality of Huatusco (a); view of sampling zones (b), the symptomatic zone A is surrounded by yellow ovals; a representative healthy or symptomatic-declining tree is shown (c, d); map (a) was created using ArcMap 10.2.2. The authors generated digital information such as polygons and contours (public domain).</p