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كتاب تسويق المحتوى الملحمي يركز على فكرة أن الشركات بحاجة إلى إنتاج محتوى مذهل يتجاوز مجرد الترويج. بدلا من إنتاج محتوى تقليدي، يركز الكتاب على فكرة إنتاج محتوى يستحق المشاركة ويساعد في بناء ثقة الجمهور. يعتبر الكتاب أن نجاح التسويق لا يتحقق عبر الطرق التقليدية (مثل الإعلانات المدفوعة) بل من خلال توفير قيمة حقيقية لجمهورك، هو دليل شامل لأي شخص أو شركة تسعى لاستخدام المحتوى كأداة قوية لبناء علاقات مستدامة مع العملاء. يعد الكتاب بمثابة دليل استراتيجي لتطوير المحتوى الملهم والمفيد الذي يحقق التأثير المطلوب على الجمهور ويسهم في النجاح على المدى الطويل.

Genome editing has emerged as a technology with a potential to revolutionize plant breeding. In this study, we report on generating, in less than ten months, Tomelo, a non-transgenic tomato variety resistant to the powdery mildew fungal pathogen using the CRISPR/Cas9 technology. We used whole-genome sequencing to show that Tomelo does not carry any foreign DNA sequences but only carries a deletion that is indistinguishable from naturally occurring mutations. We also present evidence for CRISPR/Cas9 being a highly precise tool, as we did not detect off-target mutations in Tomelo. Using our pipeline, mutations can be readily introduced into elite or locally adapted tomato varieties in less than a year with relatively minimal effort and investment.

Although genomics has become integral to life science research, inequitable access to genomics technology remains prevalent. GetGenome, a non-profit organization, aims to overcome this by providing equitable access to genomics technology and training.

Accelerated gene evolution is a hallmark of pathogen adaptation and specialization following host-jumps. However, the molecular processes associated with adaptive evolution between host-specific lineages of a multihost plant pathogen remain poorly understood. In the blast fungus Magnaporthe oryzae (Syn. Pyricularia oryzae ), host specialization on different grass hosts is generally associated with dynamic patterns of gain and loss of virulence effector genes that tend to define the distinct genetic lineages of this pathogen. Here, we unravelled the biochemical and structural basis of adaptive evolution of APikL2, an exceptionally conserved paralog of the well-studied rice-lineage specific effector AVR-Pik. Whereas AVR-Pik and other members of the six-gene AVR-Pik family show specific patterns of presence/absence polymorphisms between grass-specific lineages of M . oryzae , APikL2 stands out by being ubiquitously present in all blast fungus lineages from 13 different host species. Using biochemical, biophysical and structural biology methods, we show that a single aspartate to asparagine polymorphism expands the binding spectrum of APikL2 to host proteins of the heavy-metal associated (HMA) domain family. This mutation maps to one of the APikL2-HMA binding interfaces and contributes to an altered hydrogen-bonding network. By combining phylogenetic ancestral reconstruction with an analysis of the structural consequences of allelic diversification, we revealed a common mechanism of effector specialization in the AVR-Pik/APikL2 family that involves two major HMA-binding interfaces. Together, our findings provide a detailed molecular evolution and structural biology framework for diversification and adaptation of a fungal pathogen effector family following host-jumps.

The molecular codes underpinning the functions of plant NLR immune receptors are poorly understood. We used in vitro Mu transposition to generate a random truncation library and identify the minimal functional region of NLRs. We applied this method to NRC4—a helper NLR that functions with multiple sensor NLRs within a Solanaceae receptor network. This revealed that the NRC4 N-terminal 29 amino acids are sufficient to induce hypersensitive cell death. This region is defined by the consensus MADAxVSFxVxKLxxLLxxEx (MADA motif) that is conserved at the N-termini of NRC family proteins and ~20% of coiled-coil (CC)-type plant NLRs. The MADA motif matches the N-terminal α1 helix of Arabidopsis NLR protein ZAR1, which undergoes a conformational switch during resistosome activation. Immunoassays revealed that the MADA motif is functionally conserved across NLRs from distantly related plant species. NRC-dependent sensor NLRs lack MADA sequences indicating that this motif has degenerated in sensor NLRs over evolutionary time. Just like humans, plants get sick. They can be infected by parasites as diverse as fungi, bacteria, viruses, nematode worms and insects. But, also like humans, plants have an immune system that helps them defend against disease. Their first line of defence are disease resistance genes. Many of these genes encode so-called immune receptors, which are proteins that detect parasites and kick-off the immune response. Plant genomes may encode anywhere between 50 and 1000 immune receptors; some of which work solo as singletons, while others operate in pairs or as complex networks. Understanding how immune receptor genes have evolved would give fundamental knowledge about how they work, which in turn would set the stage for researchers to be able to use them to protect agricultural crops from disease. One driving force behind the evolution of many genes is gene duplication. Genes duplicate and afterwards the two copies can evolve in different ways. The original immune receptors are multi-tasking proteins that both detect parasites and trigger the immune response. Yet, following gene duplication, evolution has led to some immune receptors becoming dedicated to detection and losing the ability to trigger a defence response on their own. Now, Adachi et al. have discovered a molecular signature – named the MADA motif – that defines the subset of immune receptors that can trigger the immune response in plants. This motif is made of just 21 amino acids (the building blocks of proteins) at one end of the receptor and, remarkably, a short fragment of the protein containing this motif is enough to trigger a defence response when produced in plants. In contrast, the immune receptors that have specialized to only detect parasites have lost this molecular signature throughout evolution, presumably because they do not need it as they rely on their receptor partners to trigger defences instead. Every year, billions of dollars’ worth of food is lost to plant diseases. These new findings will enable the research community to classify disease resistance genes into categories to help deduce the network architecture of the plant immune system. A better understanding of this, and how networks of plant immune receptor evolve, should set the stage for breeding crop plants that are more able to resist diseases.

Supernumerary mini-chromosomes–a unique type of genomic structural variation–have been implicated in the emergence of virulence traits in plant pathogenic fungi. However, the mechanisms that facilitate the emergence and maintenance of mini-chromosomes across fungi remain poorly understood. In the blast fungus Magnaporthe oryzae (Syn. Pyricularia oryzae ), mini-chromosomes have been first described in the early 1990s but, until very recently, have been overlooked in genomic studies. Here we investigated structural variation in four isolates of the blast fungus M . oryzae from different grass hosts and analyzed the sequences of mini-chromosomes in the rice, foxtail millet and goosegrass isolates. The mini-chromosomes of these isolates turned out to be highly diverse with distinct sequence composition. They are enriched in repetitive elements and have lower gene density than core-chromosomes. We identified several virulence-related genes in the mini-chromosome of the rice isolate, including the virulence-related polyketide synthase Ace1 and two variants of the effector gene AVR-Pik . Macrosynteny analyses around these loci revealed structural rearrangements, including inter-chromosomal translocations between core- and mini-chromosomes. Our findings provide evidence that mini-chromosomes emerge from structural rearrangements and segmental duplication of core-chromosomes and might contribute to adaptive evolution of the blast fungus.

Background Understanding the mechanisms and timescales of plant pathogen outbreaks requires a detailed genome-scale analysis of their population history. The fungus Magnaporthe (Syn. Pyricularia ) oryzae —the causal agent of blast disease of cereals— is among the most destructive plant pathogens to world agriculture and a major threat to the production of rice, wheat, and other cereals. Although M. oryzae is a multihost pathogen that infects more than 50 species of cereals and grasses, all rice-infecting isolates belong to a single genetically defined lineage. Here, we combined the two largest genomic datasets to reconstruct the genetic history of the rice-infecting lineage of M. oryzae based on 131 isolates from 21 countries. Results The global population of the rice blast fungus consists mainly of three well-defined genetic groups and a diverse set of individuals. Multiple population genetic tests revealed that the rice-infecting lineage of the blast fungus probably originated from a recombining diverse group in Southeast Asia followed by three independent clonal expansions that took place over the last ~ 200 years. Patterns of allele sharing identified a subpopulation from the recombining diverse group that introgressed with one of the clonal lineages before its global expansion. Remarkably, the four genetic lineages of the rice blast fungus vary in the number and patterns of presence and absence of candidate effector genes. These genes encode secreted proteins that modulate plant defense and allow pathogen colonization. In particular, clonal lineages carry a reduced repertoire of effector genes compared with the diverse group, and specific combinations of presence and absence of effector genes define each of the pandemic clonal lineages. Conclusions Our analyses reconstruct the genetic history of the rice-infecting lineage of M. oryzae revealing three clonal lineages associated with rice blast pandemics. Each of these lineages displays a specific pattern of presence and absence of effector genes that may have shaped their adaptation to the rice host and their evolutionary history.

Background Phytophthora infestans is the most devastating pathogen of potato and a model organism for the oomycetes. It exhibits high evolutionary potential and rapidly adapts to host plants. The P. infestans genome experienced a repeat-driven expansion relative to the genomes of Phytophthora sojae and Phytophthora ramorum and shows a discontinuous distribution of gene density. Effector genes, such as members of the RXLR and Crinkler (CRN) families, localize to expanded, repeat-rich and gene-sparse regions of the genome. This distinct genomic environment is thought to contribute to genome plasticity and host adaptation. Results We used in silico approaches to predict and describe the repertoire of P. infestans secreted proteins (the secretome). We defined the \"plastic secretome\" as a subset of the genome that (i) encodes predicted secreted proteins, (ii) is excluded from genome segments orthologous to the P. sojae and P. ramorum genomes and (iii) is encoded by genes residing in gene sparse regions of P. infestans genome. Although including only ~3% of P. infestans genes, the plastic secretome contains ~62% of known effector genes and shows >2 fold enrichment in genes induced in planta . We highlight 19 plastic secretome genes induced in planta but distinct from previously described effectors. This list includes a trypsin-like serine protease, secreted oxidoreductases, small cysteine-rich proteins and repeat containing proteins that we propose to be novel candidate virulence factors. Conclusions This work revealed a remarkably diverse plastic secretome. It illustrates the value of combining genome architecture with comparative genomics to identify novel candidate virulence factors from pathogen genomes.

Aphids are amongst the most devastating sap-feeding insects of plants. Like most plant parasites, aphids require intimate associations with their host plants to gain access to nutrients. Aphid feeding induces responses such as clogging of phloem sieve elements and callose formation, which are suppressed by unknown molecules, probably proteins, in aphid saliva. Therefore, it is likely that aphids, like plant pathogens, deliver proteins (effectors) inside their hosts to modulate host cell processes, suppress plant defenses, and promote infestation. We exploited publicly available aphid salivary gland expressed sequence tags (ESTs) to apply a functional genomics approach for identification of candidate effectors from Myzus persicae (green peach aphid), based on common features of plant pathogen effectors. A total of 48 effector candidates were identified, cloned, and subjected to transient overexpression in Nicotiana benthamiana to assay for elicitation of a phenotype, suppression of the Pathogen-Associated Molecular Pattern (PAMP)-mediated oxidative burst, and effects on aphid reproductive performance. We identified one candidate effector, Mp10, which specifically induced chlorosis and local cell death in N. benthamiana and conferred avirulence to recombinant Potato virus X (PVX) expressing Mp10, PVX-Mp10, in N. tabacum, indicating that this protein may trigger plant defenses. The ubiquitin-ligase associated protein SGT1 was required for the Mp10-mediated chlorosis response in N. benthamiana. Mp10 also suppressed the oxidative burst induced by flg22, but not by chitin. Aphid fecundity assays revealed that in planta overexpression of Mp10 and Mp42 reduced aphid fecundity, whereas another effector candidate, MpC002, enhanced aphid fecundity. Thus, these results suggest that, although Mp10 suppresses flg22-triggered immunity, it triggers a defense response, resulting in an overall decrease in aphid performance in the fecundity assays. Overall, we identified aphid salivary proteins that share features with plant pathogen effectors and therefore may function as aphid effectors by perturbing host cellular processes.

Nuclei of arbuscular endomycorrhizal fungi have been described as highly diverse due to their asexual nature and absence of a single cell stage with only one nucleus. This has raised fundamental questions concerning speciation, selection and transmission of the genetic make-up to next generations. Although this concept has become textbook knowledge, it is only based on studying a few loci, including 45S rDNA. To provide a more comprehensive insight into the genetic makeup of arbuscular endomycorrhizal fungi, we applied de novo genome sequencing of individual nuclei of Rhizophagus irregularis. This revealed a surprisingly low level of polymorphism between nuclei. In contrast, within a nucleus, the 45S rDNA repeat unit turned out to be highly diverged. This finding demystifies a long-lasting hypothesis on the complex genetic makeup of arbuscular endomycorrhizal fungi. Subsequent genome assembly resulted in the first draft reference genome sequence of an arbuscular endomycorrhizal fungus. Its length is 141 Mbps, representing over 27,000 protein-coding gene models. We used the genomic sequence to reinvestigate the phylogenetic relationships of Rhizophagus irregularis with other fungal phyla. This unambiguously demonstrated that Glomeromycota are more closely related to Mucoromycotina than to its postulated sister Dikarya.