Comparative proteomic analysis identifies proteins associated with arbuscular mycorrhizal symbiosis in Poncirus trifoliata.

Citrus, one of the most widely cultivated fruit crops in the world, relies on arbuscular mycorrhizal fungi (AMF) to absorb nutrients and water from soil. However, the molecular mechanism of AM symbiosis (AMS) in citrus in general have largely been understudied. Here, using a TMT labeling proteomic approach, we identified 365 differentially expressed proteins (DEPs) in roots of Poncirus trifoliata (a common citrus rootstock) upon Rhizophagus irregularis colonization as compared with uninoculated roots, of which 287 were up-regulated and 78 were down-regulated. GO analysis revealed that the DEPs were mainly involved in biological processes such as negative regulation of endopeptidase inhibitor activity, negative regulation of endopeptidase, one-carbon metabolic process and carbohydrate metabolic process. KEGG enrichment analysis indicated that the DEPs were mainly involved in regulating metabolic pathways such as fatty acid biosynthesis, phenylpropanoid biosynthesis and carbon metabolism. Furthermore, 194 of the 365 DEPs were found to be associated with AMS-responsive genes by association analysis with our previous transcriptomes data, which highlighted the important roles of these proteins in AMS. One of the 194 DEPs, neutral ceramidase (PtNCER), was further chosen for function analysis via RNAi interfering its homologous gene MtNCER in a mycorrhizal model plant Medicago truncatula, which confirmed a positive role of NCER in AM establishment. Our results provided basic data and key candidate genes for genetic improvement of efficient nutrient uptake through AM establishment in citrus and other crops.

Plant lysin motif extracellular proteins are required for arbuscular mycorrhizal symbiosis.

Arbuscular mycorrhizal fungi (AMF) can form a mutually beneficial symbiotic relationship with most land plants. They are known to secrete lysin motif (LysM) effectors into host root cells for successful colonization. Intriguingly, plants secrete similar types of LysM proteins; however, their role in plant-microbe interactions is unknown. Here, we show that Medicago truncatula deploys LysM extracellular (LysMe) proteins to facilitate symbiosis with AMF. Promoter analyses demonstrated that three M. truncatula LysMe genes MtLysMe1/2/3, are expressed in arbuscule-containing cells and those adjacent to intercellular hyphae. Localization studies showed that these proteins are targeted to the periarbuscular space between the periarbuscular membrane and the fungal cell wall of the branched arbuscule. M. truncatula mutants in which MtLysMe2 was knocked out via CRISPR/Cas9-targeted mutagenesis exhibited a significant reduction in AMF colonization and arbuscule formation, whereas genetically complemented transgenic plants restored wild-type level AMF colonization. In addition, knocking out the ortholog of MtLysMe2 in tomato resulted in a similar defect in AMF colonization. In vitro binding affinity precipitation assays suggested binding of MtLysMe1/2/3 with chitin and chitosan, while microscale thermophoresis (MST) assays revealed weak binding of these proteins with chitooligosaccharides. Moreover, application of purified MtLysMe proteins to root segments could suppress chitooctaose (CO8)-induced reactive oxygen species production and expression of reporter genes of the immune response without impairing chitotetraose (CO4)-triggered symbiotic responses. Taken together, our results reveal that plants, like their fungal partners, also secrete LysM proteins to facilitate symbiosis establishment.

Integrated miRNA-mRNA analysis reveals candidate miRNA family regulating arbuscular mycorrhizal symbiosis of Poncirus trifoliata.

Over 70% land plants live in mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi, and maintenance of symbiosis requires transcriptional and post-transcriptional regulation. The former has been widely studied, whereas the latter mediated by symbiotic microRNAs (miRNAs) remains obscure, especially in woody plants. Here, we performed high-throughput sequencing of the perennial woody citrus plant Poncirus trifoliata and identified 3750 differentially expressed genes (DEGs) and 42 miRNAs (DEmiRs) upon AM fungal colonization. By analyzing cis-regulatory elements in the promoters of the DEGs, we predicted 329 key AM transcription factors (TFs). A miRNA-mRNA regulatory network was then constructed by integrating these data. Several candidate miRNA families of P. trifoliata were identified whose members target known symbiotic genes, such as miR167h-AMT2;3 and miR156e-EXO70I, or key TFs, such as miR164d-NAC and miR477a-GRAS, thus are involved in AM symbiotic processes of fungal colonization, arbuscule development, nutrient exchange and phytohormone signaling. Finally, analysis of selected miRNA family revealed that a miR159b conserved in mycorrhizal plant species and a Poncirus-specific miR477a regulate AM symbiosis. The role of miR477a was likely to target GRAS family gene RAD1 in citrus plants. Our results not only revealed that miRNA-mRNA network analysis, especially miRNA-TF analysis, is effective in identifying miRNA family regulating AM symbiosis, but also shed light on miRNA-mediated post-transcriptional regulation of AM symbiosis in woody citrus plants.

Cytological Observation of the Infectious Process of Venturia carpophila on Peach Leaves.

Peach scab caused by Venturia carpophila is one of the most destructive fungal diseases of peach worldwide, and it seriously affects peach production. Until now,the infectious process and pathogenesis of V. carpophila on peach have remained unclear. Here we present the infection behavior of V. carpophila at the ultrastructural and cytological levels in peach leaves with combined microscopic investigations (i.e., light microscopy, confocal laser scanning microscopy, scanning electron microscopy, and transmission electron microscopy). V. carpophila germinated at the tip of conidia and produced short germ tubes on peach leaf surfaces at 2 days post inoculation (dpi). At 3 dpi, swollen tips of germ tubes differentiated into appressoria. At 5 dpi, penetration pegs produced by appressoria broke through the cuticle layer and then differentiated into thick subcuticular hyphae in the pectin layer of the epidermal cell walls. At 10 dpi, the subcuticular hyphae extensively colonized in the pectin layer. The primary hyphae ramified into secondary hyphae and proliferated along with the incubation. At 15 dpi, the subcuticular hyphae divided laterally to form stromata between the cuticle layer and the cellulose layer of the epidermal cells. At 30 dpi, conidiophores developed from the subcuticular stromata. Finally, abundant conidiophores and new conidia appeared on leaf surfaces at 40 dpi. These results provide useful information for further a understanding of V. carpophila pathogenesis.

A Medicago truncatula SWEET transporter implicated in arbuscule maintenance during arbuscular mycorrhizal symbiosis.

Plants form a mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi, which facilitates the acquisition of scarce minerals from the soil. In return, the host plants provide sugars and lipids to its fungal partner. However, the mechanism by which the AM fungi obtain sugars from the plant has remained elusive. In this study we investigated the role of potential SWEET family sugar exporters in AM symbiosis in Medicago truncatula. We show that M. truncatula SWEET1b transporter is strongly upregulated in arbuscule-containing cells compared to roots and localizes to the peri-arbuscular membrane, across which nutrient exchange takes place. Heterologous expression of MtSWEET1b in a yeast hexose transport mutant showed that it mainly transports glucose. Overexpression of MtSWEET1b in M. truncatula roots promoted the growth of intraradical mycelium during AM symbiosis. Surprisingly, two independent Mtsweet1b mutants, which are predicted to produce truncated protein variants impaired in glucose transport, exhibited no significant defects in AM symbiosis. However, arbuscule-specific overexpression of MtSWEET1bY57A/G58D , which are considered to act in a dominant-negative manner, resulted in enhanced collapse of arbuscules. Taken together, our results reveal a (redundant) role for MtSWEET1b in the transport of glucose across the peri-arbuscular membrane to maintain arbuscules for a healthy mutually beneficial symbiosis.

Comparative transcriptome analysis of Poncirus trifoliata identifies a core set of genes involved in arbuscular mycorrhizal symbiosis.

The perennial woody plants of citrus are one of the most important fruit crops in the world and largely depends on arbuscular mycorrhizal symbiosis (AMS) to obtain essential nutrients from soil. However, the molecular aspects of AMS in citrus and perennial woody plants in general have largely been understudied. We used RNA-sequencing to identify differentially expressed genes in roots of Poncirus trifoliata upon mycorrhization by the AM fungus Glomus versiforme and evaluated their conservation by comparative transcriptome analyses with four herbaceous model plants. We identified 282 differentially expressed genes in P. trifoliata, including orthologs of 21 genes with characterized roles in AMS and 83 genes that are considered to be conserved in AM-host plants. Comparative transcriptome analysis revealed a 'core set' of 156 genes from P. trifoliata whose orthologous genes from at least three of the five species also exhibited similar transcriptional changes during AMS. Functional analysis of one of these conserved AM-induced genes, a 3-keto-acyl-ACP reductase (FatG) involved in fatty acid biosynthesis, confirmed its involvement in AMS in Medicago truncatula. Our results identify a core transcriptional program for AMS that is largely conserved between P. trifoliata and other plants. The comparative transcriptomics approach adds to previous phylogenomics studies to identify conserved genes required for AMS.