Chromosome-Scale Genome Sequence of Alternaria alternata Causing Alternaria Brown Spot of Citrus.

Alternaria brown spot (ABS), caused by Alternaria alternata, is an economically important fungal disease of citrus worldwide. The ABS pathogen A. alternata tangerine pathotype can produce a host-specific ACT toxin, which is regulated by ACT toxin gene cluster located in the conditionally dispensable chromosome (CDC). Previously, we have assembled a draft genome of A. alternata tangerine pathotype strain Z7, which comprises 165 contigs. In this study, we report a chromosome-level genome assembly of A. alternata Z7 through the combination of Oxford Nanopore sequencing and Illumina sequencing technologies. The assembly of A. alternata Z7 had a total size of 34.28 Mb, with a GC content of 51.01% and contig N50 of 3.08 Mb. The genome is encompassed 12,067 protein-coding genes, 34 ribosomal RNAs, and 107 transfer RNAs. Interestingly, A. alternata Z7 is composed of 10 essential chromosomes and 2 CDCs, which is consistent with the experimental evidences of pulsed-field gel electrophoresis. To our best knowledge, this is the first chromosome-level genome assembly of A. alternata. In addition, a database for citrus-related Alternaria genomes has been established to provide public resources for the sequences, annotation and comparative genomics data of Alternaria spp. The improved genome sequence and annotation at the chromosome level is a significant step toward a better understanding of the pathogenicity of A. alternata. The database will be updated regularly whenever the genomes of newly isolated Alternaria spp. are available. The citrus-related Alternaria genomes database is open accessible through the Citrus Fungal Disease Database.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The critical role of MetR/MetB/MetC/MetX in cysteine and methionine metabolism, fungal development and virulence of Alternaria alternata.

Methionine is a unique sulfur-containing amino acid, which plays an important role in biological protein synthesis and various cellular processes. Here, we characterized the biological functions of AaMetB, AaMetC, and AaMetX in the tangerine pathotype of Alternaria alternata Morphological analysis showed that the mutants lacking AaMetB, AaMetC, or AaMetX resulted in less aerial hypha and fewer conidia in artificial media. Pathogenicity analysis showed that AaMetB, AaMetC, and AaMetX are required for full virulence. The defects in vegetative growth, conidiation and virulence of ΔMetB, ΔMetC, and ΔMetX can be restored by exogenous methionine and homocysteine, indicating that AaMetB, AaMetC, and AaMetX are required for methionine biosynthesis. However, exogenous cysteine only restored the growth and virulence defects of ΔMetR but not ΔMetB/C/X, suggesting that AaMetR is essential for cysteine biosynthesis. Oxidant sensitivity assay showed that only ΔMetR is sensitive to H2O2 and many ROS-generating compounds, indicating that AaMetR is essential for oxidative tolerance. Interestingly, fungicides indoor bioassays showed that only the ΔMetR mutants are susceptive to chlorothalonil, a fungicide that could bind to the cysteine of glyceraldehyde-3-phosphate dehydrogenase. Comparative transcriptome analysis showed that the inactivation of MetB, MetC, MetX, or MetR significantly affected the expression of methionine metabolism-related genes. Moreover, the inactivation of AaMetR significantly affected the expression of many genes related to glutathione metabolism, which is essential for ROS tolerance. Taken together, our study provides genetic evidence to define the critical roles of AaMetB, AaMetC, AaMetX, and AaMetR in cysteine and methionine metabolism, fungal development and virulence of Alternaria alternata IMPORTANCE The transcription factor METR regulating methionine metabolism is essential for reactive oxygen species (ROS) tolerance and virulence in many phytopathogenic fungi. However, the underlying regulatory mechanism of METR involved in this process is still unclear. In the present study, we generated AaMetB, AaMetC and AaMetX deletion mutants and compared these mutants with AaMetR disrupted mutants. Interestingly, we found that AaMetB, AaMetC and AaMetX are required for vegetative growth, conidiation, and pathogenicity in Alternaria alternata, but not for ROS tolerance and cysteine metabolism. Furthermore, we found that METR is involved in the biosynthesis of cysteine, which is an essential substrate for the biosynthesis of methionine and glutathione. This study emphasizes the critical roles of MetR, MetB, MetC, MetX in the regulation of cysteine and methionine metabolism, as well as the cross-link with glutathione-mediated ROS tolerance in phytopathogenic fungi, which provides a foundation for future investigations.

The Genome Sequence of the Citrus Melanose Pathogen Diaporthe citri and Two Citrus-Related Diaporthe Species.

Melanose disease is one the most widely distributed and economically important fungal diseases of citrus worldwide. The causative agent is the filamentous fungus Diaporthe citri (syn. Phomopsis citri). Here, we report the genome assemblies of three strains of D. citri, namely strains ZJUD2, ZJUD14, and Q7, which were generated using a combination of PacBio Sequel long-read and Illumina paired-end sequencing data. The assembled genomes of D. citri ranged from 52.06 to 63.61 Mb in genome size, containing 15,977 to 16,622 protein-coding genes. We also sequenced and annotated the genome sequences of two citrus-related Diaporthe species, D. citriasiana and D. citrichinensis. In addition, a database for citrus-related Diaporthe genomes was established to provide a public platform to access genome sequences, genome annotation and comparative genomics data of these Diaporthe species. The described genome sequences and the citrus-related Diaporthe genomes database provide a useful resource for the study of fungal biology, pathogen-host interaction, molecular diagnostic marker development, and population genomic analyses of Diaporthe species. The database will be updated regularly when the genomes of newly isolated Diaporthe species are sequenced. The citrus-related Diaporthe genomes database is freely available for nonprofit use at zjudata.com/blast/diaporthe.php.

Deep Sequencing of the Medicago truncatula Root Transcriptome Reveals a Massive and Early Interaction between Nodulation Factor and Ethylene Signals.

The legume-rhizobium symbiosis is initiated through the activation of the Nodulation (Nod) factor-signaling cascade, leading to a rapid reprogramming of host cell developmental pathways. In this work, we combine transcriptome sequencing with molecular genetics and network analysis to quantify and categorize the transcriptional changes occurring in roots of Medicago truncatula from minutes to days after inoculation with Sinorhizobium medicae. To identify the nature of the inductive and regulatory cues, we employed mutants with absent or decreased Nod factor sensitivities (i.e. Nodulation factor perception and Lysine motif domain-containing receptor-like kinase3, respectively) and an ethylene (ET)-insensitive, Nod factor-hypersensitive mutant (sickle). This unique data set encompasses nine time points, allowing observation of the symbiotic regulation of diverse biological processes with high temporal resolution. Among the many outputs of the study is the early Nod factor-induced, ET-regulated expression of ET signaling and biosynthesis genes. Coupled with the observation of massive transcriptional derepression in the ET-insensitive background, these results suggest that Nod factor signaling activates ET production to attenuate its own signal. Promoter:ß-glucuronidase fusions report ET biosynthesis both in root hairs responding to rhizobium as well as in meristematic tissue during nodule organogenesis and growth, indicating that ET signaling functions at multiple developmental stages during symbiosis. In addition, we identified thousands of novel candidate genes undergoing Nod factor-dependent, ET-regulated expression. We leveraged the power of this large data set to model Nod factor- and ET-regulated signaling networks using MERLIN, a regulatory network inference algorithm. These analyses predict key nodes regulating the biological process impacted by Nod factor perception. We have made these results available to the research community through a searchable online resource.

Symbiotic rhizobia bacteria trigger a change in localization and dynamics of the Medicago truncatula receptor kinase LYK3.

To form nitrogen-fixing symbioses, legume plants recognize a bacterial signal, Nod Factor (NF). The legume Medicago truncatula has two predicted NF receptors that direct separate downstream responses to its symbiont Sinorhizobium meliloti. NOD FACTOR PERCEPTION encodes a putative low-stringency receptor that is responsible for calcium spiking and transcriptional responses. LYSIN MOTIF RECEPTOR-LIKE KINASE3 (LYK3) encodes a putative high-stringency receptor that mediates bacterial infection. We localized green fluorescent protein (GFP)-tagged LYK3 in M. truncatula and found that it has a punctate distribution at the cell periphery consistent with a plasma membrane or membrane-tethered vesicle localization. In buffer-treated control roots, LYK3:GFP puncta are dynamic. After inoculation with compatible S. meliloti, LYK3:GFP puncta are relatively stable. We show that increased LYK3:GFP stability depends on bacterial NF and NF structure but that NF is not sufficient for the change in LYK3:GFP dynamics. In uninoculated root hairs, LYK3:GFP has little codistribution with mCherry-tagged FLOTILLIN4 (FLOT4), another punctate plasma membrane-associated protein required for infection. In inoculated root hairs, we observed an increase in FLOT4:mCherry and LYK3:GFP colocalization; both proteins localize to positionally stable puncta. We also demonstrate that the localization of tagged FLOT4 is altered in plants carrying a mutation that inactivates the kinase domain of LYK3. Our work indicates that LYK3 protein localization and dynamics are altered in response to symbiotic bacteria.

Development of tools for the biochemical characterization of the symbiotic receptor-like kinase DMI2.

The Medicago truncatula DMI2 gene encodes a leucine-rich repeat receptor-like kinase that is essential for symbiosis with nitrogen-fixing rhizobia. While phenotypic analyses have provided a description for the host's responses mediated by DMI2, a lack of tools for in vivo biochemical analysis has hampered efforts to elucidate the mechanisms by which DMI2 mediates symbiotic signal transduction. Here, we report stably transformed M. truncatula lines that express a genomic DMI2 construct that is fused to a dual-affinity tag containing three copies of the hemagglutinin epitope and a single StrepII tag (gDMI2:HAST). gDMI2: HAST complements the dmi2-1 mutation, and transgenic plants expressing this construct behave similarly to wild-type plants. We show that the expression patterns of gDMI2:HAST recapitulate those of endogenous DMI2 and that we can detect and purify DMI2:HAST from microsomal root and nodule extracts. Using this line, we show that DMI2 resides in a high-molecular weight complex, which is consistent with our observation that DMI2:GFP localizes to plasma membrane-associated puncta and cytoplasmic vesicles. We further demonstrate that Nod factor (NF) perception increases the abundance of DMI2 vesicles. These tools should be a valuable resource for the Medicago community to dissect the biochemical function of DMI2.

Identification of legume RopGEF gene families and characterization of a Medicago truncatula RopGEF mediating polar growth of root hairs.

Root hairs play important roles in the interaction of plants with their environment. Root hairs anchor the plant in the soil, facilitate nutrient uptake from the rhizosphere, and participate in symbiotic plant-microbe interactions. These specialized cells grow in a polar fashion which gives rise to their elongated shape, a process mediated in part by a family of small GTPases known as Rops. RopGEFs (GEF, guanine nucleotide exchange factor) activate Rops to effect tip growth in Arabidopsis pollen and root hairs, but the genes mediating tip growth in legumes have not yet been characterized. In this report we describe the Rop and RopGEF gene families from the model legume Medicago truncatula and from the crop legume soybean. We find that one member of the M. truncatula gene family, MtRopGEF2, is required for root hair development because silencing this gene by RNA interference affects the cytosolic Ca2+ gradient and subcellular structure of root hairs, and reduces root hair growth. Consistent with its role in polar growth, we find that a GFP::MtRopGEF2 fusion protein localizes in the apex of emerging and actively growing root hairs. The amino terminus of MtRopGEF2 regulates its ability to interact with MtRops in yeast, and regulates its biological activity in vivo.

Genomic diversity and distribution of Mesorhizobium nodulating chickpea (Cicer arietinum L.) from low pH soils of Ethiopia.

Chickpea is the third most important grain legume worldwide. This is due in part to its high protein content that results from its ability to acquire bioavailable nitrogen when colonized by diverse, nitrogen fixing Mesorhizobium species. However, the diversity and distribution of mesorhizobia communities may depend on their adaptation to soil conditions. Therefore, this study was initiated in order to isolate and investigate the diversity and taxonomic identities of chickpea-nodulating Mesorhizobium species from low pH soils of Ethiopia. A total of 81 rhizobia strains were isolated from chickpea nodules harvested from low pH soils throughout Ethiopia, and their genomes were sequenced and assembled. Considering a representative set of the best-sequenced 81 genomes, the average sequence depth was 30X, with estimated average genome sizes of approximately 7 Mbp. Annotation of the assembled genome predicted an average of 7,453 protein-coding genes. Concatenation of 400 universal PhyloPhlAn conserved genes present in the genomes of all 81 strains allowed detailed phylogenetic analysis, from which eight well-supported species were identified, including M.opportunistum, M.australicum, Mesorhizobium sp. LSJC280BOO, M.wenxiniae, M.amorphae, M.loti and M.plurifarium, as well as a novel species. Phylogenetic reconstructions based on the symbiosis-related (nodC and nifH) genes were different from the core genes and consistent with horizontal transfer of the symbiotic island. The two major genomic groups, M.plurifarium and M.loti, were widely distributed in almost all the sites. The geographic pattern of genomic diversity indicated there was no relationship between geographic and genetic distance (r = 0.01, p > 0.01). In conclusion, low pH soils in Ethiopia harbored a diverse group of Mesorhizobium species, several of which were not previously known to nodulate chickpea.

Tracing nonlegume orthologs of legume genes required for nodulation and arbuscular mycorrhizal symbioses.

Most land plants can form a root symbiosis with arbuscular mycorrhizal (AM) fungi for assimilation of inorganic phosphate from the soil. In contrast, the nitrogen-fixing root nodule symbiosis is almost completely restricted to the legumes. The finding that the two symbioses share common signaling components in legumes suggests that the evolutionarily younger nitrogen-fixing symbiosis has recruited functions from the more ancient AM symbiosis. The recent advances in cloning of the genes required for nodulation and AM symbioses from the two model legumes, Medicago truncatula and Lotus japonicus, provide a unique opportunity to address biological questions pertaining to the evolution of root symbioses in plants. Here, we report that nearly all cloned legume genes required for nodulation and AM symbioses have their putative orthologs in nonlegumes. The orthologous relationship can be clearly defined on the basis of both sequence similarity and microsyntenic relationship. The results presented here serve as a prelude to the comparative analysis of orthologous gene function between legumes and nonlegumes and facilitate our understanding of how gene functions and signaling pathways have evolved to generate species- or family-specific phenotypes.

Genetic and genomic analysis in model legumes bring Nod-factor signaling to center stage.

The control of host-specificity in the Rhizobium-legume symbiosis has been a topic of long-standing interest to plant biologists. By the early 1990s, biologists had deciphered the chemical signals that trigger early symbiotic responses. Flavonoids from the plant root trigger bacterial gene expression and the production of lipo-chitooligosaccharide signals (called Nod factors) that are recognized by the plant host. Genetic differences between bacterial strains modify the oligosaccharide backbone, for example by the addition of sulfate, acetate or fucose, and simultaneously alter the host-specificity of the purified Nod factor and the bacterium. Recent studies have begun to reveal the genetic and molecular basis of Nod-factor perception in legumes, a signaling system that also controls plant interactions with mycorrhizal fungi.

The symbiotic ion channel homolog DMI1 is localized in the nuclear membrane of Medicago truncatula roots.

Legumes utilize a common signaling pathway to form symbiotic associations both with rhizobial bacteria and arbuscular mycorrhizal fungi. The perception of microbial signals is believed to take place at the plasma membrane, activating a cascade that converges on the nucleus where transcriptional reprogramming facilitates the symbioses. Forward genetic strategies have identified genes in this signaling pathway including Medicago truncatula DMI1 (Doesn't Make Infections 1) that encodes a putative ion channel. Although the DMI1 homologs from Lotus japonicus, CASTOR and POLLUX, were recently reported to be localized in plastids, we report here that a functional DMI1::GFP fusion is localized to the nuclear envelope in M. truncatula roots when expressed both from a constitutive 35S promoter and from a native DMI1 promoter. Localization may be mediated in part by sequences located within the amino-terminus of DMI1. This region of DMI1 is required for symbiotic signal transduction, and its replacement with a bona fide plastid transit peptide from the glutamine synthetase 2 gene does not restore DMI1 function. These new data place DMI1 in the nuclear envelope in close proximity to the origin of Nod-factor-induced calcium spiking.

The Medicago truncatula DMI1 protein modulates cytosolic calcium signaling.

In addition to establishing symbiotic relationships with arbuscular mycorrhizal fungi, legumes also enter into a nitrogen-fixing symbiosis with rhizobial bacteria that results in the formation of root nodules. Several genes involved in the development of both arbuscular mycorrhiza and legume nodulation have been cloned in model legumes. Among them, Medicago truncatula DMI1 (DOESN'T MAKE INFECTIONS1) is required for the generation of nucleus-associated calcium spikes in response to the rhizobial signaling molecule Nod factor. DMI1 encodes a membrane protein with striking similarities to the Methanobacterium thermoautotrophicum potassium channel (MthK). The cytosolic C terminus of DMI1 contains a RCK (regulator of the conductance of K(+)) domain that in MthK acts as a calcium-regulated gating ring controlling the activity of the channel. Here we show that a dmi1 mutant lacking the entire C terminus acts as a dominant-negative allele interfering with the formation of nitrogen-fixing nodules and abolishing the induction of calcium spikes by the G-protein agonist Mastoparan. Using both the full-length DMI1 and this dominant-negative mutant protein we show that DMI1 increases the sensitivity of a sodium- and lithium-hypersensitive yeast (Saccharomyces cerevisiae) mutant toward those ions and that the C-terminal domain plays a central role in regulating this response. We also show that DMI1 greatly reduces the release of calcium from internal stores in yeast, while the dominant-negative allele appears to have the opposite effect. This work suggests that DMI1 is not directly responsible for Nod factor-induced calcium changes, but does have the capacity to regulate calcium channels in both yeast and plants.

Medicago truncatula DMI1 required for bacterial and fungal symbioses in legumes.

Legumes form symbiotic associations with both mycorrhizal fungi and nitrogen-fixing soil bacteria called rhizobia. Several of the plant genes required for transduction of rhizobial signals, the Nod factors, are also necessary for mycorrhizal symbiosis. Here, we describe the cloning and characterization of one such gene from the legume Medicago truncatula. The DMI1 (does not make infections) gene encodes a novel protein with low global similarity to a ligand-gated cation channel domain of archaea. The protein is highly conserved in angiosperms and ancestral to land plants. We suggest that DMI1 represents an ancient plant-specific innovation, potentially enabling mycorrhizal associations.