Host Susceptibility Genes Research Articles

A triple threat: the Parastagonospora nodorum SnTox267 effector exploits three distinct host genetic factors to cause disease in wheat.

Summary Parastagonospora nodorum is a fungal pathogen of wheat. As a necrotrophic specialist, it deploys effector proteins that target dominant host susceptibility genes to elicit programmed cell death (PCD). Here we identify and functionally validate the effector targeting the host susceptibility genes Snn2, Snn6 and Snn7.We utilized whole‐genome sequencing, association mapping, gene‐disrupted mutants, gain‐of‐function transformants, virulence assays, bioinformatics and quantitative PCR to characterize these interactions.A single proteinaceous effector, SnTox267, targeted Snn2, Snn6 and Snn7 to trigger PCD. Snn2 and Snn6 functioned cooperatively to trigger PCD in a light‐dependent pathway, whereas Snn7‐mediated PCD functioned in a light‐independent pathway. Isolates harboring 20 SnTox267 protein isoforms quantitatively varied in virulence. The diversity and distribution of isoforms varied between populations, indicating adaptation to local selection pressures. SnTox267 deletion resulted in the upregulation of effector genes SnToxA, SnTox1 and SnTox3.We validated a novel effector operating in an inverse‐gene‐for‐gene manner to target three genetically distinct host susceptibility genes and elicit PCD. The discovery of the complementary gene action of Snn2 and Snn6 indicates their potential function in a guard or decoy model. Additionally, differences in light dependency in the elicited pathways and upregulation of unlinked effectors sheds new light onto a complex fungal necrotroph–host interaction.

An accumulation of genetic variation and selection across the disease-related genes during apple domestication

Although human-imposed selection is known to have altered plant traits during crop domestication, the effect of selection on host susceptibility or resistance is not well understood. Moreover, the domestication of perennial tree fruit crops, unlike annual crops, was driven by hybridization, clonal propagation, and selection of desirable phenotypes, which could confound the accurate assessment of domestication-associated effects on host resistance or susceptibility genes. We studied the effects of domestication on disease-related nucleotide-binding leucine-rich repeat (NLR) genes by combining phylogenetic, haplotype, and selection signature analysis using sequence data from Golden Delicious double haploid (GDDH13 v.1.1) apple genome and 80 resequenced domesticated and wild Malus accessions. The NLR gene family in the GDDH13 v.1.1 apple genome constituted 546 genes that showed expansion mainly through proximal (39.1%) and dispersed (29.5%) duplications. The genome duplication (WGD) within Rosaceae, affecting the Malus lineage, is also evident in the NLR genes. The NLR genes are found in genomic regions associated with previously detected disease resistance-related quantitative trait loci (QTL) for apple scab, fire blight, powdery mildew, and blue mold. A genomic diversity analysis identified that the NLR genes in domesticated apples (Malus domestica) have more variation (average πdom=4.01 × 10−3) than their main progenitor, wild M. sieversii (average πsie=2.95 × 10−3) and M. sylvestris (average πsyl=2.11 × 10−3), which hybridized with M. domestica during the domestication process. These results suggest evidence of selection on disease-related genes associated with domestication of apple. An increased diversity across NLR genes in the domesticated germplasm may be attributed to their diverse geographical origins and distinct pedigrees, together with selection for disease resistance during domestication. The NLR genes under selection can provide opportunities to explore their role in disease resistance in apples.

Long-Read Genome Sequence Resources of Xanthomonas citri pv. punicae Strain Bagalkot Causing Pomegranate Bacterial Blight.

Xanthomonas citri pv. punicae causing bacterial blight is a devastating disease of pomegranate in India and Pakistan. Most xanthomonads use the type III secretion system to inject transcription activator-like effector (TALE) proteins into the host cell. TALEs bind to the effector-binding elements in the promoter of host susceptibility genes, triggering disease development. PacBio single-molecule real-time long-read sequencing technology was used to identify the TALE-encoding genes, which is otherwise not possible using next-generation short-read sequencers. In all, 1.74 Gb of raw data containing 368,980 subreads, with an average read length of 4,724 bp and longest read length of 77,471, were generated. Subreads were assembled into 15 scaffolds generating approximately 5.4 Mb (348×) of genome. X. citri pv. punicae exhibited close lineage with X. citri pv. citri with 98.78% average nucleotide identity. Of the 4,263 protein-coding genes, 11 non-TALE type III effectors and 2 TALE-encoding genes were identified.[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.

Consequences of adaptation of TAL effectors on host susceptibility to Xanthomonas.

Transcription activator-like effectors (TALEs) are virulence factors of Xanthomonas that induce the expression of host susceptibility (S) genes by specifically binding to effector binding elements (EBEs) in their promoter regions. The DNA binding specificity of TALEs is dictated by their tandem repeat regions, which are highly variable between different TALEs. Mutation of the EBEs of S genes is being utilized as a key strategy to generate resistant crops against TALE-dependent pathogens. However, TALE adaptations through rearrangement of their repeat regions is a potential obstacle for successful implementation of this strategy. We investigated the consequences of TALE adaptations in the citrus pathogen Xanthomonas citri subsp. citri (Xcc), in which PthA4 is the TALE required for pathogenicity, whereas CsLOB1 is the corresponding susceptibility gene, on host resistance. Seven TALEs, containing two-to-nine mismatching-repeats to the EBEPthA4 that were unable to induce CsLOB1 expression, were introduced into Xcc pthA4:Tn5 and adaptation was simulated by repeated inoculations into and isolations from sweet orange for a duration of 30 cycles. While initially all strains failed to promote disease, symptoms started to appear between 9-28 passages in four TALEs, which originally harbored two-to-five mismatches. Sequence analysis of adapted TALEs identified deletions and mutations within the TALE repeat regions which enhanced putative affinity to the CsLOB1 promoter. Sequence analyses suggest that TALEs adaptations result from recombinations between repeats of the TALEs. Reintroduction of these adapted TALEs into Xcc pthA4:Tn5 restored the ability to induce the expression of CsLOB1, promote disease symptoms and colonize host plants. TALEs harboring seven-to-nine mismatches were unable to adapt to overcome the incompatible interaction. Our study experimentally documented TALE adaptations to incompatible EBE and provided strategic guidance for generation of disease resistant crops against TALE-dependent pathogens.

RNA-Based Technologies for Engineering Plant Virus Resistance.

In recent years, non-coding RNAs (ncRNAs) have gained unprecedented attention as new and crucial players in the regulation of numerous cellular processes and disease responses. In this review, we describe how diverse ncRNAs, including both small RNAs and long ncRNAs, may be used to engineer resistance against plant viruses. We discuss how double-stranded RNAs and small RNAs, such as artificial microRNAs and trans-acting small interfering RNAs, either produced in transgenic plants or delivered exogenously to non-transgenic plants, may constitute powerful RNA interference (RNAi)-based technology that can be exploited to control plant viruses. Additionally, we describe how RNA guided CRISPR-CAS gene-editing systems have been deployed to inhibit plant virus infections, and we provide a comparative analysis of RNAi approaches and CRISPR-Cas technology. The two main strategies for engineering virus resistance are also discussed, including direct targeting of viral DNA or RNA, or inactivation of plant host susceptibility genes. We also elaborate on the challenges that need to be overcome before such technologies can be broadly exploited for crop protection against viruses.

Control of Plant Viruses by CRISPR/Cas System-Mediated Adaptive Immunity.

Plant diseases caused by invading plant viruses pose serious threats to agricultural production in the world, and the antiviral engineering initiated by molecular biotechnology has been an effective strategy to prevent and control plant viruses. Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system-mediated DNA or RNA editing/interference in plants make them very attractive tools applicable to the plant protection field. Here, we review the development of CRISPR/Cas systems and summarize their applications in controlling different plant viruses by targeting viral sequences or host susceptibility genes. We list some potential recessive resistance genes that can be utilized in antiviral breeding and emphasize the importance and promise of recessive resistance gene-based antiviral breeding to generate transgene-free plants without developmental defects. Finally, we discuss the challenges and opportunities for the application of CRISPR/Cas techniques in the prevention and control of plant viruses in the field.

Co-Expression of Mitochondrial Genes and ACE2 in Cornea Involved in COVID-19.

PurposeThe coronavirus disease 2019 (COVID-19) pandemic severely challenges public health and necessitates the need for increasing our understanding of COVID-19 pathogenesis, especially host factors facilitating virus infection and propagation. The aim of this study was to investigate key factors for cellular susceptibility to severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection in the ocular surface cells.MethodsWe combined co-expression and SARS-CoV-2 interactome network to predict key genes at COVID-19 in ocular infection based on the premise that genes underlying a disease are often functionally related and functionally related genes are often co-expressed.ResultsThe co-expression network was constructed by mapping the well-known angiotensin converting enzyme (ACE2), TMPRSS2, and host susceptibility genes implicated in COVID-19 genomewide association study (GWAS) onto a cornea, retinal pigment epithelium, and lung. We found a significant co-expression module of these genes in the cornea, revealing that cornea is potential extra-respiratory entry portal of SARS-CoV-2. Strikingly, both co-expression and interaction networks show a significant enrichment in mitochondrial function, which are the hub of cellular oxidative homeostasis, inflammation, and innate immune response. We identified a corneal mitochondrial susceptibility module (CMSM) of 14 mitochondrial genes by integrating ACE2 co-expression cluster and SARS-CoV-2 interactome. The gene ECSIT, as a cytosolic adaptor protein involved in inflammatory responses, exhibits the strongest correlation with ACE2 in CMSM, which has shown to be an important risk factor for SARS-CoV-2 infection and prognosis.ConclusionsOur co-expression and protein interaction network analysis uncover that the mitochondrial function related genes in cornea contribute to the dissection of COVID-19 susceptibility and potential therapeutic interventions.

The immunity of Meiwa kumquat against Xanthomonas citri is associated with a known susceptibility gene induced by a transcription activator-like effector.

Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is one of the most devastating diseases in citrus. Meiwa kumquat (Fortunella crassifolia) has shown a durable resistance against Xcc. Here, we aimed to characterize the mechanisms responsible for such a durable resistance by characterizing the transcriptional and physiological responses of Meiwa kumquat to Xcc. Inoculation of Meiwa kumquat with Xcc promoted immune responses such as upregulation of PR genes, accumulation of salicylic acid, hypersensitive response (HR)-like cell death and early leaf abscission. Hypertrophy and hyperplasia symptoms, which are known to be caused by Xcc-induction of the canker susceptibility gene LOB1 through the transcription activator-like effector (TALE) PthA4, always appear prior to the development of cell death. Mutation of pthA4 in Xcc abolished the induction of LOB1, canker symptoms, cell death, and leaf abscission and reduced the expression of PR genes in inoculated kumquat leaves without reducing Xcc titers in planta. Transcriptome analysis demonstrated that PthA4 promotes plant biotic and abiotic stress responses and the biosynthesis of abscisic acid. Transcriptional induction of LOB1 homologs in Meiwa kumquat by Xcc pthA4 mutant strains carrying a repertoire of designer TALEs promoted the elicitation of HR-like phenotype and leaf abscission, suggesting that kumquat response to Xcc is associated with upregulation of LOB1. Our study suggests a novel mechanism of plant resistance to Xanthomonas via elicitation of immune responses by upregulation of a host susceptibility gene.

Cost-Effective and Time-Efficient Molecular Assisted Selection for PPV Resistance in Apricot Based on ParPMC2 Allele-Specific PCR

Plum pox virus (PPV) is the most important limiting factor for apricot (Prunus armeniaca L.) production worldwide, and development of resistant cultivars has been proven to be the best solution in the long-term. However, just like in other woody species, apricot breeding is highly time and space demanding, and this is particularly true for PPV resistance phenotyping. Therefore, marker-assisted selection (MAS) may be very helpful to speed up breeding programs. Tightly linked ParPMC1 and ParPMC2, meprin and TRAF-C homology (MATH)-domain-containing genes have been proposed as host susceptibility genes required for PPV infection. Contribution of additional genes to PPV resistance cannot be discarded, but all available studies undoubtedly show a strong correlation between ParPMC2-resistant alleles (ParPMC2res) and PPV resistance. The ParPMC2res allele was shown to carry a 5-bp deletion (ParPMC2-del) within the second exon that has been characterized as a molecular marker suitable for MAS (PMC2). Based on this finding, we propose here a method for PPV resistance selection in apricot by combining high-throughput DNA extraction of 384 samples in 2 working days and the allele-specific genotyping of PMC2 on agarose gel. Moreover, the PMC2 genotype has been determined by PCR or by using whole-genome sequences (WGS) in 175 apricot accessions. These results were complemented with phenotypic and/or genotypic data available in the literature to reach a total of 325 apricot accessions. As a whole, we conclude that this is a time-efficient, cost-effective and straightforward method for PPV resistance screening that can be highly useful for apricot breeding programs.

A Loss-of-Function Mutation in the Integrin Alpha L (Itgal) Gene Contributes to Susceptibility to Salmonella enterica Serovar Typhimurium Infection in Collaborative Cross Strain CC042.

Salmonella is an intracellular bacterium found in the gastrointestinal tract of mammalian, avian, and reptilian hosts. Mouse models have been extensively used to model in vivo distinct aspects of human Salmonella infections and have led to the identification of several host susceptibility genes. We have investigated the susceptibility of Collaborative Cross strains to intravenous infection with Salmonella enterica serovar Typhimurium as a model of human systemic invasive infection. In this model, strain CC042/GeniUnc (CC042) mice displayed extreme susceptibility with very high bacterial loads and mortality. CC042 mice showed lower spleen weights and decreased splenocyte numbers before and after infection, affecting mostly CD8+ T cells, B cells, and all myeloid cell populations, compared with control C57BL/6J mice. CC042 mice also had lower thymus weights with a reduced total number of thymocytes and double-negative and double-positive (CD4+, CD8+) thymocytes compared to C57BL/6J mice. Analysis of bone marrow-resident hematopoietic progenitors showed a strong bias against lymphoid-primed multipotent progenitors. An F2 cross between CC042 and C57BL/6N mice identified two loci on chromosome 7 (Stsl6 and Stsl7) associated with differences in bacterial loads. In the Stsl7 region, CC042 carried a loss-of-function variant, unique to this strain, in the integrin alpha L (Itgal) gene, the causative role of which was confirmed by a quantitative complementation test. Notably, Itgal loss of function increased the susceptibility to S. Typhimurium in a (C57BL/6J × CC042)F1 mouse background but not in a C57BL/6J mouse inbred background. These results further emphasize the utility of the Collaborative Cross to identify new host genetic variants controlling susceptibility to infections and improve our understanding of the function of the Itgal gene.

Effect of the host-specific toxin SnTOX3 from Stagonospora nodorum on ethylene signaling pathway regulation and redox-state in common wheat

The fungus Stagonospora nodorum Berk. is the causative agent of Septoria nodorum blotch (SNB) of wheat. The most important factors of Stagonospora nodorum virulence include numerous fungal necrotrophic effectors (NEs) encoded by SnTox genes. They interact with the matching products of host susceptibility genes (Snn). SnTox-Snn interactions are mirror images of classical gene-for-gene interactions and lead to the development of disease. We have studied the SnTox3-Snn3 interaction, resulting in the development of infection on leaves and formation of extensive lesions. The mechanism of SnTox3 action is likely to be linked to the regulation of redox metabolism and the influence on ethylene synthesis in the wheat plants, although the molecular mechanisms are not fully unveiled. To characterize the SnTox3-Snn3 interaction, we used S. nodorum isolates differing in the expression of the NEs genes SnTox3 (SnB (Tox3+), Sn4VD (Tox3–)) and two soft spring wheat (Triticum aestivum L.) cultivars, contrasting in resistance to the SNB agent and differing in the allelic composition of the susceptibility locus Snn3-B1: Kazakhstanskaya 10 (susceptible) and Omskaya 35 (resistant). We carried out a comparative assessment of the transcriptional activity patterns of genes responsible for ethylene biosynthesis (TaACS1, TaACО) and signaling pathway (TaEIL1, TaERF1) by real-time PCR and estimated the redox state of wheat plants infected with different isolates of S. nodorum by spectrometry. The induction of ethylene biosynthesis and signaling has been shown to result from gene-for-gene interaction between Snn3-B1 and SnTox3. The results of plant redox status estimation showed that ethylene inhibited accumulation of hydrogen peroxide in SnTox3-sensitive genotypes by regulating the operation of various pro-/antioxidant enzymes at the transcriptional and posttranslational levels. Our results suggest that NE SnTox3 influences ethylene biosynthesis and signaling, thereby regulating redox metabolism in infected wheat plants as necessary for successful host colonization at the initial phases of infection, which ultimately leads to extensive lesions due to fast pathogen reproduction.

CRISPR Applications in Plant Virology: Virus Resistance and Beyond.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated genes (Cas) is a prokaryotic adaptive immune system which has been reprogrammed into a precise, simple, and efficient gene targeting technology. This emerging technology is revolutionizing various areas of life sciences, medicine, and biotechnology and has raised significant interest among plant biologists, both in basic science and in plant protection and breeding. In this review, we describe the basic principles of CRISPR/Cas systems, and how they can be deployed to model plants and crops for the control, monitoring, and study of the mechanistic aspects of plant virus infections. We discuss how Cas endonucleases can be used to engineer plant virus resistance by directly targeting viral DNA or RNA, as well as how they can inactivate host susceptibility genes. Additionally, other applications of CRISPR/Cas in plant virology such as virus diagnostics and imaging are reviewed. The review also provides a systemic comparison between CRISPR/Cas technology and RNA interference approaches, the latter of which has also been used for development of virus-resistant plants. Finally, we outline challenges to be solved before CRISPR/Cas can produce virus-resistant crop plants which can be marketed.

Using in vitro plants to study the cassava response to Xanthomonas phaseoli pv. manihotis infection

Cassava Bacterial Blight (CBB), caused by Xanthomonas phaseoli pv. manihotis (Xpm), is the most important bacterial disease of cassava (Manihot esculenta). Xpm employs Transcription Activator-Like effectors (TALE) to induce the expression of host susceptibility (S) genes favoring bacterial growth. The identification of resistant and susceptible cassava varieties as well as the identification of the cassava gene MeSweet10a as a target of the effector TAL20, have been conducted on plants grown from mature stem-cuttings. We explored the possibility of using in vitro plants to study cassava-Xpm interactions. Cassava in vitro plants of varieties 60444 and CM6438–14 were susceptible and resistant, respectively, to infection by strain Xpm668. In addition, the expression of MeSweet10a was induced in the susceptible but not in the resistant variety, which was not associated with polymorphisms in the Effector Binding Element (EBE) reported for TAL20. Three other Xpm strains, Xpm531, Xpm681 and Xpm1061, were also able to induce the TAL20 cognate target gene in the susceptible cassava variety 60444. These results demonstrate the usefulness of in vitro plants to study the phenotypic and molecular responses of cassava during Xpm infection.

Current status on mapping of genes for resistance to leaf- and neck-blast disease in rice.

Blast disease caused by fungal pathogen Pyricularia oryzae is a major threat to rice productivity worldwide. The rice-blast pathogen can infect both leaves and panicle neck nodes. Nearly, 118 genes for resistance to leaf blast have been identified and 25 of these have been molecularly characterized. A great majority of these genes encode nucleotide-binding site-leucine-rich repeat (NBS-LRR) proteins and are organized into clusters as allelic or tightly linked genes. Compared to ever expanding list of leaf-blast-resistance genes, a few major genes mediating protection to neck blast have been identified. A great majority of the genetic studies conducted with the genotypes differing in the degree of susceptibility/resistance to neck blast have suggested quantitative inheritance for the trait. Several reports on co-localization of gene/QTLs for leaf- and neck-blast resistance in rice genome have suggested the existence of common genes for resistance to both phases of the disease albeit inconsistencies in the genomic positions leaf- and neck-blast-resistance genes in some instances have presented the contrasting scenario. There is a strong evidence to suggest that developmentally regulated expression of many blast-resistance genes is a key determinant deciding their effectiveness against leaf or neck blast. Testing of currently characterized leaf-blast-resistance genes for their reaction to neck blast is required to expand the existing repertoire resistance genes against neck blast. Current developments in the understanding of molecular basis of host-pathogen interactions in rice-blast pathosystem offer novel possibilities for achieving durable resistance to blast through exploitation of natural or genetically engineered loss-of-function alleles of host susceptibility genes.

Harnessing Genome Editing Techniques to Engineer Disease Resistance in Plants.

Modern genome editing (GE) techniques, which include clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system, transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs) and LAGLIDADG homing endonucleases (meganucleases), have so far been used for engineering disease resistance in crops. The use of GE technologies has grown very rapidly in recent years with numerous examples of targeted mutagenesis in crop plants, including gene knockouts, knockdowns, modifications, and the repression and activation of target genes. CRISPR/Cas9 supersedes all other GE techniques including TALENs and ZFNs for editing genes owing to its unprecedented efficiency, relative simplicity and low risk of off-target effects. Broad-spectrum disease resistance has been engineered in crops by GE of either specific host-susceptibility genes (S gene approach), or cleaving DNA of phytopathogens (bacteria, virus or fungi) to inhibit their proliferation. This review focuses on different GE techniques that can potentially be used to boost molecular immunity and resistance against different phytopathogens in crops, ultimately leading to the development of promising disease-resistant crop varieties.

The host basal transcription factor IIA subunits coordinate for facilitating infection of TALEs-carrying bacterial pathogens in rice

Many plant-pathogenic Xanthomonas rely largely on secreting virulence transcription activator-like effectors (TALEs) proteins into plant nucleus to activate host susceptibility genes to cause disease, the process is dependent on pathogen TALEs association with host plants basal transcription factor IIA small subunit TFIIAγ. TFIIAγ together with large subunit TFIIAαβ constitute as a key component of RNA polymerase II complex for transcriptome initiation. However, whether TFIIAαβ coordinates or competes with pathogen TALEs for interaction with TFIIAγ to activate transcript of TALEs-targeting genes is unclear. Here, we showed that TALE-carrying bacterial pathogens Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), the causal agent for bacterial leaf blight and bacterial leaf streak in rice, using their major virulence TALEs to physically associate with N-terminal of OsTFIIAγ5. OsTFIIAα and OsTFIIAβ which are post-translationally mature proteins of OsTFIIAαβ separately bound to N- and C-terminal of OsTFIIAγ5. OsTFIIAα coordinated with TALEs for binding with OsTFIIAγ5 to upregulate rice susceptibility genes to cause disease. Conversely, suppression of OsTFIIAαβ attenuated TALEs-targeting genes transcription, thus improved broad-spectrum disease resistance of rice to Xoo and Xoc. These results provide an applicable strategy for improving resistance to TALE-carrying pathogens in rice by appropriate suppression of plant basal transcription factors expression.

Microbial network disturbances in relapsing refractory Crohn's disease.

Inflammatory bowel diseases (IBD) can be broadly divided into Crohn's disease (CD) and ulcerative colitis (UC) from their clinical phenotypes. Over 150 host susceptibility genes have been described, although most overlap between CD, UC and their subtypes, and they do not adequately account for the overall incidence or the highly variable severity of disease. Replicating key findings between two long-term IBD cohorts, we have defined distinct networks of taxa associations within intestinal biopsies of CD and UC patients. Disturbances in an association network containing taxa of the Lachnospiraceae and Ruminococcaceae families, typically producing short chain fatty acids, characterize frequently relapsing disease and poor responses to treatment with anti-TNF-α therapeutic antibodies. Alterations of taxa within this network also characterize risk of later disease recurrence of patients in remission after the active inflamed segment of CD has been surgically removed.

CRISPR/Cas9: A Novel Weapon in the Arsenal to Combat Plant Diseases.

Plant pathogens like virus, bacteria, and fungi incur a huge loss of global productivity. Targeting the dominant R gene resulted in the evolution of resistance in pathogens, which shifted plant pathologists’ attention toward host susceptibility factors (or S genes). Herein, the application of sequence-specific nucleases (SSNs) for targeted genome editing are gaining more importance, which utilize the use of meganucleases (MN), zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN) with the latest one namely clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). The first generation of genome editing technologies, due to their cumbersome nature, is becoming obsolete. Owing to its simple and inexpensive nature the use of CRISPR/Cas9 system has revolutionized targeted genome editing technology. CRISPR/Cas9 system has been exploited for developing resistance against virus, bacteria, and fungi. For resistance to DNA viruses (mainly single-stranded DNA viruses), different parts of the viral genome have been targeted transiently and by the development of transgenic plants. For RNA viruses, mainly the host susceptibility factors and very recently the viral RNA genome itself have been targeted. Fungal and bacterial resistance has been achieved mainly by targeting the host susceptibility genes through the development of transgenics. In spite of these successes CRISPR/Cas9 system suffers from off-targeting. This and other problems associated with this system are being tackled by the continuous discovery/evolution of new variants. Finally, the regulatory standpoint regarding CRISPR/Cas9 will determine the fate of using this versatile tool in developing pathogen resistance in crop plants.

Dictyostelium Host Response to Legionella Infection: Strategies and Assays.

The professional phagocyte Dictyostelium discoideum is a well-established model organism to study host-pathogen interactions. Dictyostelium amoebae grow as separate, independent cells; they divide by binary fission and take up bacteria and yeast via phagocytosis. In the year 2000, D. discoideum was described by two groups as a novel system for genetic analysis of host-pathogen interactions for the intracellular pathogen Legionella pneumophila. Since then additional microbial pathogens that can be studied in D. discoideum have been reported. The organism has various advantages for the dissection of the complex cross-talk between a host and a pathogen. A fully sequenced and well-curated genome is available, there are excellent molecular genetic tools on the market, and the generation of targeted multiple gene knock-outs as well as the realization of untargeted genetic screens is generally straightforward. Dictyostelium also offers easy cultivation, and the cells are suitable for cell biological studies, which in combination with in vivo expression of fluorescence-tagged proteins allows the investigation of the dynamics of bacterial uptake and infection. Furthermore, a large mutant collection is available at the Dictyostelium stock center, favoring the identification of host resistance or susceptibility genes. Here, we briefly describe strategies to identify host cell factors important during an infection, followed by protocols for cell culture and storage, uptake and infection, and confocal microscopy of infected cells.

Identification and characterization of GLDC as host susceptibility gene to severe influenza

Glycine decarboxylase (GLDC) was prioritized as a candidate susceptibility gene to severe influenza in humans. The higher expression of GLDC derived from genetic variations may confer a higher risk to H7N9 and severe H1N1 infection. We sought to characterize GLDC as functional susceptibility gene that GLDC may intrinsically regulate antiviral response, thereby impacting viral replication and disease outcome. We demonstrated that GLDC inhibitor AOAA and siRNA depletion boosted IFNβ‐ and IFN‐stimulated genes (ISGs) in combination with PolyI:C stimulation. GLDC inhibition and depletion significantly amplified antiviral response of type I IFNs and ISGs upon viral infection and suppressed the replication of H1N1 and H7N9 viruses. Consistently, GLDC overexpression significantly promoted viral replication due to the attenuated antiviral responses. Moreover, GLDC inhibition in H1N1‐infected BALB/c mice recapitulated the amplified antiviral response and suppressed viral growth. AOAA provided potent protection to the infected mice from lethal infection, comparable to a standard antiviral against influenza viruses. Collectively, GLDC regulates cellular antiviral response and orchestrates viral growth. GLDC is a functional susceptibility gene to severe influenza in humans.