State‐of‐the‐art CRISPR/Cas9 Technology for Genome Editing in Trypanosomatids

CRISPR/Cas9 technology has revolutionized biology. This prokaryotic defense system against foreign DNA has been repurposed for genome editing in a broad range of cell tissues and organisms. Trypanosomatids are flagellated protozoa belonging to the order Kinetoplastida. Some of its most representative members cause important human diseases affecting millions of people worldwide, such as Chagas disease, sleeping sickness and different forms of leishmaniases. Trypanosomatid infections represent an enormous burden for public health and there are no effective treatments for most of the diseases they cause. Since the emergence of the CRISPR/Cas9 technology, the genetic manipulation of these parasites has notably improved. As a consequence, genome editing is now playing a key role in the functional study of proteins, in the characterization of metabolic pathways, in the validation of alternative targets for antiparasitic interventions, and in the study of parasite biology and pathogenesis. In this work we review the different strategies that have been used to adapt the CRISPR/Cas9 system to Trypanosoma cruzi, Trypanosoma brucei, and Leishmania spp., as well as the research progress achieved using these approaches. Thereby, we will present the state‐of‐the‐art molecular tools available for genome editing in trypanosomatids to finally point out the future perspectives in the field.     

Population structure and reticulate evolution of Saccharomyces eubayanus and its lager‐brewing hybrids

Reticulate evolution can be a major driver of diversification into new niches, especially in disturbed habitats and at the edges of ranges. Industrial fermentation strains of yeast provide a window into these processes, but progress has been hampered by a limited understanding of the natural diversity and distribution of Saccharomyces species and populations. For example, lager beer is brewed with Saccharomyces pastorianus, an alloploid hybrid of S. cerevisiae and S. eubayanus, a species only recently discovered in Patagonia, Argentina. Here, we report that genetically diverse strains of S. eubayanus are readily isolated from Patagonia, demonstrating that the species is well established there. Analyses of multilocus sequence data strongly suggest that there are two diverse and highly differentiated Patagonian populations. The low nucleotide diversity found in the S. eubayanus moiety of hybrid European brewing strains suggests that their alleles were drawn from a small subpopulation that is closely related to one of the Patagonian populations. For the first time, we also report the rare isolation of S. eubayanus outside Patagonia, in Wisconsin, USA. In contrast to the clear population differentiation in Patagonia, the North American strains represent a recent and possibly transient admixture of the two Patagonian populations. These complex and varied reticulation events are not adequately captured by conventional phylogenetic methods and required analyses of Bayesian concordance factors and phylogenetic networks to accurately summarize and interpret. These findings show how genetically diverse eukaryotic microbes can produce rare but economically important hybrids with low genetic diversity when they migrate from their natural ecological context.     

Efficacy of Acibenzolar‐S‐Methyl and β‐Aminobutyric Acid for Control of Downy Mildew in Greenhouse Grown Basil and Peroxidase Activity in Response to Treatment with these Compounds

Downy mildew, caused by the oomycete pathogen Peronospora belbahrii, is a devastating foliar disease of basil in the United States and worldwide. Currently there are very few chemistries or organic choices registered to control this disease. In this study, two systemic acquired resistance (SAR) inducers, acibenzolar‐S‐methyl (ASM) and β‐aminobutyric acid (BABA), were evaluated for their in vitro effects on the pathogen, for their potential to control basil downy mildew in greenhouses, and for changes in peroxidase activity in basil plants treated with these two SAR inducers. No significant inhibition of sporangial germination was detected in water agar amended with ASM at concentrations lower than 100 mg/l or with BABA at concentrations lower than 500 mg/l. Efficacy of ASM and BABA in greenhouses varied depending on the rate, method and timing of application. The area under the disease progress curve (AUDPC) of disease severity was significantly reduced compared to the non‐treated control when ASM was sprayed (in all experiments) or drenched (in one out of two experiments) pre‐, or pre‐ + post‐inoculation at rates of 25–400 mg/l. Three weekly post‐inoculation sprays of ASM at the rate of 50 mg/l reduced AUDPC by 93.0 and 47.2% when started 3 and 7 days after inoculation (DAI), respectively. The AUDPC of disease severity was also significantly reduced when BABA was sprayed pre‐ + post‐inoculation at rates of 125–500 mg/l. According to the prediction using a log‐logistic function, 50% maximum disease protection was achieved at a concentration of 27.5 mg/l of ASM. Basil plants treated with these two SAR inducers and challenged with the pathogen showed significantly higher peroxidase activity than the non‐treated control at 8 DAI. Temporally, the highest activity of peroxidase was detected at 8 DAI, decreased at 15 DAI and waned further at 23 DAI.     

Sex‐specific responses of Asian citrus psyllid to volatiles of conspecific and host‐plant origin

Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is the primary vector of Candidatus Liberibacter spp. bacteria that cause citrus greening, a disease of worldwide importance. Olfactometry was employed to test responses of D. citri to odours from intact citrus plants (Mexican lime, Citrus aurantifolia, sour orange, Citrus aurantium, Marsh grapefruit, Citrus paradisi and Valencia orange, Citrus sinensis), citrus plants previously infested with D. citri, and odours of conspecifics including nymphs, adult insects of same and opposite sex, and their products (honeydew), both alone and in combination. In contrast to other studies, psyllids of both sexes were attracted to volatiles of undamaged Mexican lime leaves, whereas undamaged grapefruit attracted only females, and leaves of Valencia and sour orange did not attract either sex. All four plant species attracted female psyllids when previously infested, but only Mexican lime and sour orange‐attracted males. Thus, Citrus species appear to vary in the production of both constituitive and induced volatiles that attract adult psyllids. Volatiles emitted by nymphs did not attract either sex, but psyllid honeydew was attractive to males, likely due to female pheromone residues. Males oriented to the odour of females, whereas the reverse was not true, and neither males nor females oriented to same‐sex volatiles. The addition of conspecific cues (adults, nymphs or honeydew) did not increase female attraction to previously infested leaves, but male response was increased by the presence of adults and honeydew, regardless of plant species. Thus, female psyllids appear to orient more strongly to volatiles of plant origin, whereas males respond more strongly to cues emanating from females and conspecific excretions. These results suggest that female psyllids drive the initial colonization of host plants, whereas males orient to females and infested plants. Identification of the specific volatiles involved may permit their use in monitoring and management of this pest.     

Direct visualization of Agrobacterium‐delivered VirE2 in recipient cells

Agrobacterium tumefaciens is a natural genetic engineer widely used to deliver DNA into various recipients, including plant, yeast and fungal cells. The bacterium can transfer single‐stranded DNA molecules (T–DNAs) and bacterial virulence proteins, including VirE2. However, neither the DNA nor the protein molecules have ever been directly visualized after the delivery. In this report, we adopted a split‐GFP approach: the small GFP fragment (GFP11) was inserted into VirE2 at a permissive site to create the VirE2‐GFP11 fusion, which was expressed in A. tumefaciens; and the large fragment (GFP1–10) was expressed in recipient cells. Upon delivery of VirE2‐GFP11 into the recipient cells, GFP fluorescence signals were visualized. VirE2‐GFP11 was functional like VirE2; the GFP fusion movement could indicate the trafficking of Agrobacterium‐delivered VirE2. As the natural host, all plant cells seen under a microscope received the VirE2 protein in a leaf‐infiltration assay; most of VirE2 moved at a speed of 1.3–3.1 μm sec^?1 in a nearly linear direction, suggesting an active trafficking process. Inside plant cells, VirE2‐GFP formed filamentous structures of different lengths, even in the absence of T‐DNA. As a non‐natural host recipient, 51% of yeast cells received VirE2, which did not move inside yeast. All plant cells seen under a microscope transiently expressed the Agrobacterium‐delivered transgene, but only 0.2% yeast cells expressed the transgene. This indicates that Agrobacterium is a more efficient vector for protein delivery than T‐DNA transformation for a non‐natural host recipient: VirE2 trafficking is a limiting factor for the genetic transformation of a non‐natural host recipient. The split‐GFP approach could enable the real‐time visualization of VirE2 trafficking inside recipient cells.     

A dual role for integrin‐linked kinase and β1‐integrin in modulating cardiac aging

Cardiac performance decreases with age, which is a major risk factor for cardiovascular disease and mortality in the aging human population, but the molecular mechanisms underlying cardiac aging are still poorly understood. Investigating the role of integrin‐linked kinase (ilk) and β1‐integrin (myospheroid, mys) in Drosophila, which colocalize near cardiomyocyte contacts and Z‐bands, we find that reduced ilk or mys function prevents the typical changes of cardiac aging seen in wildtype, such as arrhythmias. In particular, the characteristic increase in cardiac arrhythmias with age is prevented in ilk and mys heterozygous flies with nearly identical genetic background, and they live longer, in line with previous findings in Caenorhabditis elegans for ilk and in Drosophila for mys. Consistent with these findings, we observed elevated β1‐integrin protein levels in old compared with young wild‐type flies, and cardiac‐specific overexpression of mys in young flies causes aging‐like heart dysfunction. Moreover, moderate cardiac‐specific knockdown of integrin‐linked kinase (ILK)/integrin pathway‐associated genes also prevented the decline in cardiac performance with age. In contrast, strong cardiac knockdown of ilk or ILK‐associated genes can severely compromise cardiac integrity, including cardiomyocyte adhesion and overall heart function. These data suggest that ilk/mys function is necessary for establishing and maintaining normal heart structure and function, and appropriate fine‐tuning of this pathway can retard the age‐dependent decline in cardiac performance and extend lifespan. Thus, ILK/integrin‐associated signaling emerges as an important and conserved genetic mechanism in longevity, and as a new means to improve age‐dependent cardiac performance, in addition to its vital role in maintaining cardiac integrity.     

The hybrid Four‐CBS‐Domain KINβγ subunit functions as the canonical γ subunit of the plant energy sensor SnRK1

The AMPK/SNF1/SnRK1 protein kinases are a family of ancient and highly conserved eukaryotic energy sensors that function as heterotrimeric complexes. These typically comprise catalytic α subunits and regulatory β and γ subunits, the latter function as the energy‐sensing modules of animal AMPK through adenosine nucleotide binding. The ability to monitor accurately and adapt to changing environmental conditions and energy supply is essential for optimal plant growth and survival, but mechanistic insight in the plant SnRK1 function is still limited. In addition to a family of γ‐like proteins, plants also encode a hybrid βγ protein that combines the Four‐Cystathionine β‐synthase (CBS)‐domain (FCD) structure in γ subunits with a glycogen‐binding domain (GBD), typically found in β subunits. We used integrated functional analyses by ectopic SnRK1 complex reconstitution, yeast mutant complementation, in‐depth phylogenetic reconstruction, and a seedling starvation assay to show that only the hybrid KINβγ protein that recruited the GBD around the emergence of the green chloroplast‐containing plants, acts as the canonical γ subunit required for heterotrimeric complex formation. Mutagenesis and truncation analysis further show that complex interaction in plant cells and γ subunit function in yeast depend on both a highly conserved FCD and a pre‐CBS domain, but not the GBD. In addition to novel insight into canonical AMPK/SNF/SnRK1 γ subunit function, regulation and evolution, we provide a new classification of plant FCD genes as a convenient and reliable tool to predict regulatory partners for the SnRK1 energy sensor and novel FCD gene functions.