Efficient CRISPR-mediated base editing in Agrobacterium spp.

Agrobacterium spp. are important plant pathogens that are the causative agents of crown gall or hairy root disease. Their unique infection strategy depends on the delivery of part of their DNA to plant cells. Thanks to this capacity, these phytopathogens became a powerful and indispensable tool for plant genetic engineering and agricultural biotechnology. Although Agrobacterium spp. are standard tools for plant molecular biologists, current laboratory strains have remained unchanged for decades and functional gene analysis of Agrobacterium has been hampered by time-consuming mutation strategies. Here, we developed clustered regularly interspaced short palindromic repeats (CRISPR)-mediated base editing to enable the efficient introduction of targeted point mutations into the genomes of both Agrobacterium tumefaciens and Agrobacterium rhizogenes As an example, we generated EHA105 strains with loss-of-function mutations in recA, which were fully functional for maize (Zea mays) transformation and confirmed the importance of RolB and RolC for hairy root development by A. rhizogenes K599. Our method is highly effective in 9 of 10 colonies after transformation, with edits in at least 80% of the cells. The genomes of EHA105 and K599 were resequenced, and genome-wide off-target analysis was applied to investigate the edited strains after curing of the base editor plasmid. The off-targets present were characteristic of Cas9-independent off-targeting and point to TC motifs as activity hotspots of the cytidine deaminase used. We anticipate that CRISPR-mediated base editing is the start of "engineering the engineer," leading to improved Agrobacterium strains for more efficient plant transformation and gene editing.

A Chromosome-Scale Genome Assembly Resource for Myriosclerotinia sulcatula Infecting Sedge Grass (Carex sp.).

The fungus Myriosclerotinia sulcatula is a close relative of the notorious polyphagous plant pathogens Botrytis cinerea and Sclerotinia sclerotiorum but exhibits a host range restricted to plants from the Carex genus (Cyperaceae family). To date, there are no genomic resources available for fungi in the Myriosclerotinia genus. Here, we present a chromosome-scale reference genome assembly for M. sulcatula. The assembly contains 24 contigs with a total length of 43.53 Mbp, with scaffold N50 of 2,649.7 kbp and N90 of 1,133.1 kbp. BRAKER-predicted gene models were manually curated using WebApollo, resulting in 11,275 protein-coding genes that we functionally annotated. We provide a high-quality reference genome assembly and annotation for M. sulcatula as a resource for studying evolution and pathogenicity in fungi from the Sclerotiniaceae family.

A Short-Read Genome Assembly Resource for Leveillula taurica Causing Powdery Mildew Disease of Sweet Pepper (Capsicum annuum).

Powdery mildew of sweet pepper (Capsicum annuum) is an economically important disease. It is caused by Leveillula taurica, an obligate biotrophic ascomycete with a partly endophytic mycelium and haustoria, i.e., feeding structures formed in the mesophyll cells of infected host plant tissues. The molecular basis of its pathogenesis is largely unknown because genomic resources only exist for epiphytically growing powdery mildew fungi with haustoria formed exclusively in epidermal cells of their plant hosts. Here, we present the first reference genome assembly for an isolate of L. taurica isolated from sweet pepper in Hungary. The short read-based assembly consists of 23,599 contigs with a total length of 187.2 Mbp; the scaffold N50 is 13,899 kbp and N90 is 3,522 kbp; and the average GC content is 39.2%. We detected at least 92,881 transposable elements covering 55.5 Mbp (30.4%). BRAKER predicted 19,751 protein-coding gene models in this assembly. Our reference genome assembly of L. taurica is the first resource to study the molecular pathogenesis and evolution of a powdery mildew fungus with a partly endophytic lifestyle.

Transcriptome analyses reveal SR45 to be a neutral splicing regulator and a suppressor of innate immunity in Arabidopsis thaliana.

BACKGROUND: Regulation of pre-mRNA splicing diversifies protein products and affects many biological processes. Arabidopsis thaliana Serine/Arginine-rich 45 (SR45), regulates pre-mRNA splicing by interacting with other regulatory proteins and spliceosomal subunits. Although SR45 has orthologs in diverse eukaryotes, including human RNPS1, the sr45-1 null mutant is viable. Narrow flower petals and reduced seed formation suggest that SR45 regulates genes involved in diverse processes, including reproduction. To understand how SR45 is involved in the regulation of reproductive processes, we studied mRNA from the wild-type and sr45-1 inflorescences using RNA-seq, and identified SR45-bound RNAs by immunoprecipitation. RESULTS: Using a variety of bioinformatics tools, we identified a total of 358 SR45 differentially regulated (SDR) genes, 542 SR45-dependent alternative splicing (SAS) events, and 1812 SR45-associated RNAs (SARs). There is little overlap between SDR genes and SAS genes, and neither set of genes is enriched for flower or seed development. However, transcripts from reproductive process genes are significantly overrepresented in SARs. In exploring the fate of SARs, we found that a total of 81 SARs are subject to alternative splicing, while 14 of them are known Nonsense-Mediated Decay (NMD) targets. Motifs related to GGNGG are enriched both in SARs and near different types of SAS events, suggesting that SR45 recognizes this motif directly. Genes involved in plant defense are significantly over-represented among genes whose expression is suppressed by SR45, and sr45-1 plants do indeed show enhanced immunity. CONCLUSION: We find that SR45 is a suppressor of innate immunity. We find that a single motif (GGNGG) is highly enriched in both RNAs bound by SR45 and in sequences near SR45- dependent alternative splicing events in inflorescence tissue. We find that the alternative splicing events regulated by SR45 are enriched for this motif whether the effect of SR45 is activation or repression of the particular event. Thus, our data suggests that SR45 acts to control splice site choice in a way that defies simple categorization as an activator or repressor of splicing.

Long noncoding RNAs emerge from transposon-derived antisense sequences and may contribute to infection stage-specific transposon regulation in a fungal phytopathogen.

BACKGROUND: The genome of the obligate biotrophic phytopathogenic barley powdery mildew fungus Blumeria hordei is inflated due to highly abundant and possibly active transposable elements (TEs). In the absence of the otherwise common repeat-induced point mutation transposon defense mechanism, noncoding RNAs could be key for regulating the activity of TEs and coding genes during the pathogenic life cycle. RESULTS: We performed time-course whole-transcriptome shotgun sequencing (RNA-seq) of total RNA derived from infected barley leaf epidermis at various stages of fungal pathogenesis and observed significant transcript accumulation and time point-dependent regulation of TEs in B. hordei. Using a manually curated consensus database of 344 TEs, we discovered phased small RNAs mapping to 104 consensus transposons, suggesting that RNA interference contributes significantly to their regulation. Further, we identified 5,127 long noncoding RNAs (lncRNAs) genome-wide in B. hordei, of which 823 originated from the antisense strand of a TE. Co-expression network analysis of lncRNAs, TEs, and coding genes throughout the asexual life cycle of B. hordei points at extensive positive and negative co-regulation of lncRNAs, subsets of TEs and coding genes. CONCLUSIONS: Our work suggests that similar to mammals and plants, fungal lncRNAs support the dynamic modulation of transcript levels, including TEs, during pivotal stages of host infection. The lncRNAs may support transcriptional diversity and plasticity amid loss of coding genes in powdery mildew fungi and may give rise to novel regulatory elements and virulence peptides, thus representing key drivers of rapid evolutionary adaptation to promote pathogenicity and overcome host defense.

Transcriptional response to host chemical cues underpins the expansion of host range in a fungal plant pathogen lineage.

The host range of parasites is an important factor in assessing the dynamics of disease epidemics. The evolution of pathogens to accommodate new hosts may lead to host range expansion, a process the molecular bases of which are largely enigmatic. The fungus Sclerotinia sclerotiorum has been reported to parasitize more than 400 plant species from diverse eudicot families while its close relative, S. trifoliorum, is restricted to plants from the Fabaceae family. We analyzed S. sclerotiorum global transcriptome reprogramming on hosts from six botanical families and reveal a flexible, host-specific transcriptional program. We generated a chromosome-level genome assembly for S. trifoliorum and found near-complete gene space conservation in two representative strains of broad and narrow host range Sclerotinia species. However, S. trifoliorum showed increased sensitivity to the Brassicaceae defense compound camalexin. Comparative analyses revealed a lack of transcriptional response to camalexin in the S. trifoliorum strain and suggest that regulatory variation in detoxification and effector genes at the population level may associate with the genetic accommodation of Brassicaceae in the Sclerotinia host range. Our work proposes transcriptional plasticity and the co-existence of signatures for generalist and polyspecialist adaptive strategies in the genome of a plant pathogen.

Analysis of gene expression in soybean (Glycine max) roots in response to the root knot nematode Meloidogyne incognita using microarrays and KEGG pathways.

BACKGROUND: Root-knot nematodes are sedentary endoparasites that can infect more than 3000 plant species. Root-knot nematodes cause an estimated $100 billion annual loss worldwide. For successful establishment of the root-knot nematode in its host plant, it causes dramatic morphological and physiological changes in plant cells. The expression of some plant genes is altered by the nematode as it establishes its feeding site. RESULTS: We examined the expression of soybean (Glycine max) genes in galls formed in roots by the root-knot nematode, Meloidogyne incognita, 12 days and 10 weeks after infection to understand the effects of infection of roots by M. incognita. Gene expression was monitored using the Affymetrix Soybean GeneChip containing 37,500 G. max probe sets. Gene expression patterns were integrated with biochemical pathways from the Kyoto Encyclopedia of Genes and Genomes using PAICE software. Genes encoding enzymes involved in carbohydrate and cell wall metabolism, cell cycle control and plant defense were altered. CONCLUSIONS: A number of different soybean genes were identified that were differentially expressed which provided insights into the interaction between M. incognita and soybean and into the formation and maintenance of giant cells. Some of these genes may be candidates for broadening plants resistance to root-knot nematode through over-expression or silencing and require further examination.

Post-transcriptional gene silencing of root-knot nematode in transformed soybean roots.

RNAi constructs targeted to four different genes were examined to determine their efficacy to reduce galls formed by Meloidogyne incognita in soybean roots. These genes have high similarity with essential soybean cyst nematode (Heterodera glycines) and Caenorhabditis elegans genes. Transformed roots were challenged with M. incognita. Two constructs, targeted to genes encoding tyrosine phosphatase (TP) and mitochondrial stress-70 protein precursor (MSP), respectively, strongly interfered with M. incognita gall formation. The number of galls formed on roots transformed with constructs targeting the M. incognita TP and MSP genes was reduced by 92% and 94.7%, respectively. The diameter of M. incognita inside these transformed roots was 5.4 and 6.5 times less than the diameter of M. incognita found inside control plants transformed with the empty vector. These results indicate that silencing the genes encoding TP and MSP can greatly decrease gall formation and shows a promising solution for broadening resistance of plants against this plant-parasitic nematode.

Pathogen Effectors: Exploiting the Promiscuity of Plant Signaling Hubs.

Pathogens produce effectors to overcome plant immunity, thereby threatening crop yields and global food security. Large-scale interactomic studies have revealed that pathogens from different kingdoms of life target common plant proteins during infection, the so-called effector hubs. These hubs often play central roles in numerous plant processes through their ability to interact with multiple plant proteins. This ability arises partly from the presence of intrinsically disordered domains (IDDs) in their structure. Here, we highlight the role of the TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) and JASMONATE-ZIM DOMAIN (JAZ) transcription regulator families as plant signaling and effector hubs. We consider different evolutionary hypotheses to rationalize the existence of diverse effectors sharing common targets and the possible role of IDDs in this interaction.

Genome-wide alternative splicing profiling in the fungal plant pathogen Sclerotinia sclerotiorum during the colonization of diverse host families.

Sclerotinia sclerotiorum is a notorious generalist plant pathogen that threatens more than 600 host plants, including wild and cultivated species. The molecular bases underlying the broad compatibility of S. sclerotiorum with its hosts is not fully elucidated. In contrast to higher plants and animals, alternative splicing (AS) is not well studied in plant-pathogenic fungi. AS is a common regulated cellular process that increases cell protein and RNA diversity. In this study, we annotated spliceosome genes in the genome of S. sclerotiorum and characterized their expression in vitro and during the colonization of six host species. Several spliceosome genes were differentially expressed in planta, suggesting that AS was altered during infection. Using stringent parameters, we identified 1,487 S. sclerotiorum genes differentially expressed in planta and exhibiting alternative transcripts. The most common AS events during the colonization of all plants were retained introns and the alternative 3' receiver site. We identified S. sclerotiorum genes expressed in planta for which (a) the relative accumulation of alternative transcripts varies according to the host being colonized and (b) alternative transcripts harbour distinct protein domains. This notably included 42 genes encoding predicted secreted proteins showing high-confidence AS events. This study indicates that AS events are taking place in the plant pathogenic fungus S. sclerotiorum during the colonization of host plants and could generate functional diversity in the repertoire of proteins secreted by S. sclerotiorum during infection.

Surfactin Stimulated by Pectin Molecular Patterns and Root Exudates Acts as a Key Driver of the Bacillus-Plant Mutualistic Interaction.

Bacillus velezensis is considered as a model species belonging to the so-called Bacillus subtilis complex that evolved typically to dwell in the soil rhizosphere niche and establish an intimate association with plant roots. This bacterium provides protection to its natural host against diseases and represents one of the most promising biocontrol agents. However, the molecular basis of the cross talk that this bacterium establishes with its natural host has been poorly investigated. We show here that these plant-associated bacteria have evolved a polymer-sensing system to perceive their host and that, in response, they increase the production of the surfactin-type lipopeptide. Furthermore, we demonstrate that surfactin synthesis is favored upon growth on root exudates and that this lipopeptide is a key component used by the bacterium to optimize biofilm formation, motility, and early root colonization. In this specific nutritional context, the bacterium also modulates qualitatively the pattern of surfactin homologues coproduced in planta and forms mainly variants that are the most active at triggering plant immunity. Surfactin represents a shared good as it reinforces the defensive capacity of the host. IMPORTANCE Within the plant-associated microbiome, some bacterial species are of particular interest due to the disease protective effect they provide via direct pathogen suppression and/or stimulation of host immunity. While these biocontrol mechanisms are quite well characterized, we still poorly understand the molecular basis of the cross talk these beneficial bacteria initiate with their host. Here, we show that the model species Bacillus velezensis stimulates the production of the surfactin lipopeptide upon sensing pectin as a cell surface molecular pattern and upon feeding on root exudates. Surfactin favors bacterial rhizosphere fitness on one hand and primes the plant immune system on the other hand. Our data therefore illustrate how both partners use this multifunctional compound as a unique shared good to sustain a mutualistic interaction.

Effective approaches to study the plant-root knot nematode interaction.

Plant-parasitic nematodes cause major agricultural losses worldwide. Examining the molecular mechanisms underlying plant-nematode interactions and how plants respond to different invading pathogens is attracting major attention to reduce the expanding gap between agricultural production and the needs of the growing world population. This review summarizes the most recent developments in plant-nematode interactions and the diverse approaches used to improve plant resistance against root knot nematode (RKN). We will emphasize the recent rapid advances in genome sequencing technologies, small interfering RNA techniques (RNAi) and targeted genome editing which are contributing to the significant progress in understanding the plant-nematode interaction mechanisms. Also, molecular approaches to improve plant resistance against nematodes are considered.