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.     

Interaction of MC1R and PMEL alleles on solid coat colors in Highland cattle

Six solid colors occur in Highland cattle: black, dun, silver dun and red, yellow, and white. These six coat colors are explained by a non‐epistatic interaction of the genotypes at the MC1R and PMEL genes. A three base pair deletion in the PMEL gene leading to the deletion of a leucine from the signal peptide is observed in dilute‐colored Highland cattle (c.50_52delTTC, p.Leu18del). The mutant PMEL allele acts in a semi‐dominant manner. Dun Galloway cattle also have one copy of the deletion allele, and silver dun Galloway cattle have two copies. The presence of two adjacent leucine residues at the site of this deletion is highly conserved in human, horse, mouse and chicken as well as in cattle with undiluted coat colors. Highland and Galloway cattle thus exhibit a similar dose‐dependent dilution effect based on the number of PMEL :c.50_51delTTC alleles, as Charolais cattle with PMEL :c.64G>A alleles. The PMEL :c.64G>A allele was not found in Highland or Galloway cattle.     

Light and temperature effects on photosynthetic activity of E ucheuma denticulatum and K appaphycus alvarezii (brown and green color morphotypes) from Sulawesi Utara,Indonesia

Knowledge concerning the effects of several abiotic factors on the physiology of carrageenophytes is essential both in ecological and economic standpoints, to ensure their sufficient supply for the sustainability of seaweed‐based industries. This paper presents the photosynthetic characteristics of farmed carrageenophytes, E ucheuma denticulatum and K appaphycus alvarezii [brown (BRN) and green (GRN) color morphotypes] from Sulawesi Utara (Sulawesi Island), Indonesia, as determined by examining their photosynthetic response across different temperatures and irradiances using dissolved oxygen measurements and pulse‐amplitude modulated fluorometer. Net photosynthesis–irradiance ( P – E ) curves at 26°C revealed that net photosynthetic rates of the three seaweeds gradually increased until the estimated saturation irradiances ( E _k ) of 58 μmol photons m^{? 2} s^?1 (49–68 μmol photons m^{? 2} s^?1, 95% Bayesian prediction intervals; BPI) for E . denticulatum, and 158 and 143 μmol photons m^{? 2} s^?1 (134–185 and 99–203 μmol photons m^{? 2} s^?1, 95% BPI) for BRN and GRN K . alvarezii, respectively; and that no photoinhibition was observed at the highest irradiance of 1000 μmol photons m^{? 2} s^?1. All seaweed samples exhibited photosynthetic tolerance to high PAR as shown by their recovery in maximum quantum yields (F_v / F_m ) following chronic exposures; as well as tolerance over a broad range of temperature, which is from 19 to 33°C for E . denticulatum, 20–29°C for BRN K . alvarezii, and 17–32°C for GRN K . alvarezii. Temperature responses of these carrageenophytes indicated that they were well‐adapted to the annual seawater temperatures in the cultivation site; however, they are also likely close to threshold levels for thermal inhibition, given the decline in F_v / F_m above 30°C.     

Mutant Alleles at the Brown Locus Encoding Tyrosinase‐Related Protein‐1 (TRP‐1) Affect Proliferation of Mouse Melanocytes in Culture *

Tyrosinase related protein‐1 (TRP‐1) is a melanocyte‐specific gene product involved in eumelanin synthesis. Mutation in the Tyrp1 gene is associated with brown pelage in mouse and oculocutaneous albinism Type 3 in humans (OCA3). It has been demonstrated that TRP‐1 expresses DHICA oxidase activity in the murine system. However, its actual function in the human system is still unclear. The study was designed to determine the effects of mutation at two Typr1 alleles, namely the Tyrp1^b (brown) and Tyrp1^b‐cj (cordovan) compared with wild type Tyrp1^B (black) on melanocyte function and melanin biosynthesis. The most significant finding was that both of the Tyrp1 mutations (i.e. brown expressing a point mutation and cordovan expressing decreased amount of TRP‐1 protein) resulted in attenuation of cell proliferation rates. Neither necrosis nor apoptosis was responsible for the observed decrease in cell proliferation rates of the brown and cordovan melanocytes. Ultrastructural evaluation by electron microscopic analysis revealed that both mutations in Tyrp1 affected melanosome maturation without affecting its structure. These observations demonstrate that mutation in Tyrp1 compromised tyrosinase activity within the organelle. DOPA histochemistry revealed differences in melanosomal stages between black and brown melanocytes but not between black and cordovan melanocytes. There were no significant differences in tyrosine hydroxylase activities of tyrosinase and TRP‐1 in wild type black, brown and cordovan melanocyte cell lysates. We conclude that mutations in Tyrp1 compromise cell proliferation and melanosomal maturation in mouse melanocyte cultures.     

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.     

Engineering fire blight resistance into the apple cultivar ‘Gala’ using the FB_MR5 CC‐NBS‐LRR resistance gene of Malus × robusta 5

The fire blight susceptible apple cultivar Malus × domestica Borkh. cv. ‘Gala’ was transformed with the candidate fire blight resistance gene FB_MR5 originating from the crab apple accession Malus × robusta 5 (Mr5). A total of five different transgenic lines were obtained. All transgenic lines were shown to be stably transformed and originate from different transgenic events. The transgenic lines express the FB_MR5 either driven by the constitutive CaMV 35S promoter and the ocs terminator or by its native promoter and terminator sequences. Phenotyping experiments were performed with Mr5‐virulent and Mr5‐avirulent strains of Erwinia amylovora, the causal agent of fire blight. Significantly less disease symptoms were detected on transgenic lines after inoculation with two different Mr5‐avirulent E. amylovora strains, while significantly more shoot necrosis was observed after inoculation with the Mr5‐virulent mutant strain ZYRKD3_1. The results of these experiments demonstrated the ability of a single gene isolated from the native gene pool of apple to protect a susceptible cultivar from fire blight. Furthermore, this gene is confirmed to be the resistance determinant of Mr5 as the transformed lines undergo the same gene‐for‐gene interaction in the host–pathogen relationship Mr5–E. amylovora.     

Identification and Pathogenicity of Lasiodiplodia Species from Eucalyptus urophylla × grandis,Polyscias balfouriana and Bougainvillea spectabilis in Southern China

Species of Lasiodiplodia are important pathogens of a wide variety of plants covering a wide geographical distribution. These fungi can be associated with different symptoms such as stem cankers, shoot blights, fruit rots, dieback and gummosis. Diseases caused by Lasiodiplodia were surveyed on Eucalyptus urophylla × grandis, Polyscias balfouriana and Bougainvillea spectabilis in a nursery in southern China. Based on morphology characteristics and phylogenetic analyses of ITS rDNA sequences and translation elongation factor 1‐alpha (TEF‐1α) gene regions, four species of Lasiodiplodia were identified. Lasiodiplodia theobromae was identified from E. urophylla × grandis, P. balfouriana and B. spectabilis. L. hormozganensis, L. iraniensis and L. pseudotheobromae were identified from B. spectabilis. To our knowledge, with the exception of L. theobromae on E. urophylla × grandis, this study represents the first report of these fungi on the host plants. Pathogenicity tests showed that all Lasiodiplodia spp. obtained in this study are virulent to E. urophylla × grandis and B. spectabilis, and L. theobromae was virulent to P. balfouriana.     

‘Last In–First Out’: seasonal variations of non‐structural carbohydrates,glucose‐6‐phosphate and ATP in tubers of two Arum species

• Knowledge on the metabolism of polysaccharide reserves in wild species is still scarce. In natural sites we collected tubers of Arum italicum Mill. and A. maculatum L. – two geophytes with different apparent phenological timing, ecology and chorology – during five stages of the annual cycle in order to understand patterns of reserve accumulation and degradation.
• Both the entire tuber and its proximal and distal to shoot portion were utilised. Pools of non‐structural carbohydrates (glucose, sucrose and starch), glucose‐6‐phosphate and ATP were analysed as important markers of carbohydrate metabolism.
• In both species, starch and glucose content of the whole tuber significantly increased from sprouting to the maturation/senescence stages, whereas sucrose showed an opposite trend; ATP and glucose‐6‐phosphate were almost stable and dropped only at the end of the annual cycle. Considering the two different portions of the tuber, both ATP and glucose‐6‐phosphate concentrations were higher in proximity to the shoot in all seasonal stages, except the flowering stage.
• Our findings suggest that seasonal carbon partitioning in the underground organ is driven by phenology and occurs independently of seasonal climate conditions. Moreover, our results show that starch degradation, sustained by elevated ATP and glucose‐6‐phosphate pools, starts in the peripheral, proximal‐to‐shoot portion of the tuber, consuming starch accumulated in the previous season, as a ‘Last In–First Out’ mechanism of carbohydrate storage.

     

The dorid nudibranchs Peltodoris lentiginosa and Archidoris odhneri as predators of glass sponges

The dorid nudibranchs Peltodoris lentiginosa and Archidoris odhneri were found on glass sponges (Porifera, Hexactinellida) during remotely operated vehicle surveys of three reefs in the Strait of Georgia, British Columbia, Canada. Eight nudibranchs were sampled from 2009 to 2011. Identification of sponge spicules found in their gut and fecal contents confirmed the nudibranchs to be predators of the reef‐forming hexactinellids Aphrocallistes vastus and Heterochone calyx, as well as of the demosponge Desmacella austini, which encrusts skeletons of the glass sponges. Four of five nudibranchs dissected for gut content analysis had stomachs containing sponge spicules. Counts from high‐definition video footage taken during systematic surveys done in 2009 showed that nudibranchs were found in only two of the three glass sponge reefs. These data provide the first quantitative evidence of a molluscan predator on glass sponges found outside of Antarctica, and establish the first trophic link between glass sponges and their associated community of animals in a sponge reef ecosystem on the western Canadian continental shelf.     

Functional analysis of endo‐1,4‐β‐glucanases in response to Botrytis cinerea and Pseudomonas syringae reveals their involvement in plant–pathogen interactions

Plant cell wall modification is a critical component in stress responses. Endo‐1,4‐β‐glucanases (EGs) take part in cell wall editing processes, e.g. elongation, ripening and abscission. Here we studied the infection response of Solanum lycopersicum and Arabidopsis thaliana with impaired EGs. Transgenic TomCel1 and TomCel2 tomato antisense plants challenged with Pseudomonas syringae showed higher susceptibility, callose priming and increased jasmonic acid pathway marker gene expression. These two EGs could be resistance factors and may act as negative regulators of callose deposition, probably by interfering with the defence‐signalling network. A study of a set of Arabidopsis EG T‐DNA insertion mutants challenged with P. syringae and Botrytis cinerea revealed that the lack of other EGs interferes with infection phenotype, callose deposition, expression of signalling pathway marker genes and hormonal balance. We conclude that a lack of EGs could alter plant response to pathogens by modifying the properties of the cell wall and/or interfering with signalling pathways, contributing to generate the appropriate signalling outcomes. Analysis of microarray data demonstrates that EGs are differentially expressed upon many different plant–pathogen challenges, hormone treatments and many abiotic stresses. We found some Arabidopsis EG mutants with increased tolerance to osmotic and salt stress. Our results show that impairing EGs can alter plant–pathogen interactions and may contribute to appropriate signalling outcomes in many different biotic and abiotic plant stress responses.     

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.     

Amplicon‐pyrosequencing‐based detection of compositional shifts in bryophyte‐associated fungal communities along an elevation gradient

Although bryophytes are a dominant vegetation component of boreal and alpine ecosystems, little is known about their associated fungal communities. HPLC assays of ergosterol (fungal biomass) and amplicon pyrosequencing of the ITS2 region of rDNA were used to investigate how the fungal communities associated with four bryophyte species changed across an elevational gradient transitioning from conifer forest to the low‐alpine. Fungal biomass and OTU richness associated with the four moss hosts did not vary significantly across the gradient (P > 0.05), and both were more strongly affected by host and tissue type. Despite largely constant levels of fungal biomass, distinct shifts in community composition of fungi associated with Hylocomium, Pleurozium and Polytrichum occurred between the elevation zones of the gradient. This likely is a result of influence on fungal communities by major environmental factors such as temperature, directly or indirectly mediated by, or interacting with, the response of other components of the vegetation (i.e. the dominant trees). Fungal communities associated with Dicranum were an exception, exhibiting spatial autocorrelation between plots, and no significant structuring by elevation. Nevertheless, the detection of distinct fungal assemblages associated with a single host growing in different elevation zones along an elevational gradient is of particular relevance in the light of the ongoing changes in vegetation patterns in boreal and alpine systems due to global climate warming.     

HT proteins contribute to S‐RNase‐independent pollen rejection in Solanum

Plants have mechanisms to recognize and reject pollen from other species. Although widespread, these mechanisms are less well understood than the self‐incompatibility (SI) mechanisms plants use to reject pollen from close relatives. Previous studies have shown that some interspecific reproductive barriers (IRBs) are related to SI in the Solanaceae. For example, the pistil SI proteins S‐RNase and HT protein function in a pistil‐side IRB that causes rejection of pollen from self‐compatible (SC) red/orange‐fruited species in the tomato clade. However, S‐RNase‐independent IRBs also clearly contribute to rejecting pollen from these species. We investigated S‐RNase‐independent rejection of Solanum lycopersicum pollen by SC Solanum pennellii LA0716, SC. Solanum habrochaites LA0407, and SC Solanum arcanum LA2157, which lack functional S‐RNase expression. We found that all three accessions express HT proteins, which previously had been known to function only in conjunction with S‐RNase, and then used RNAi to test whether they also function in S‐RNase‐independent pollen rejection. Suppressing HT expression in SC S. pennellii LA0716 allows S. lycopersicum pollen tubes to penetrate farther into the pistil in HT suppressed plants, but not to reach the ovary. In contrast, suppressing HT expression in SC. Solanum habrochaites LA0407 and in SC S. arcanum LA2157 allows S. lycopersicum pollen tubes to penetrate to the ovary and produce hybrids that, otherwise, would be difficult to obtain. Thus, HT proteins are implicated in both S‐RNase‐dependent and S‐RNase‐independent pollen rejection. The results support the view that overall compatibility results from multiple pollen–pistil interactions with additive effects.     

Cofeeding intra‐ and interspecific transmission of an emerging insect‐borne rickettsial pathogen

Cat fleas (Ctenocephalides felis) are known as the primary vector and reservoir of Rickettsia felis, the causative agent of flea‐borne spotted fever; however, field surveys regularly report molecular detection of this infectious agent from other blood‐feeding arthropods. The presence of R. felis in additional arthropods may be the result of chance consumption of an infectious bloodmeal, but isolation of viable rickettsiae circulating in the blood of suspected vertebrate reservoirs has not been demonstrated. Successful transmission of pathogens between actively blood‐feeding arthropods in the absence of a disseminated vertebrate infection has been verified, referred to as cofeeding transmission. Therefore, the principal route from systemically infected vertebrates to uninfected arthropods may not be applicable to the R. felis transmission cycle. Here, we show both intra‐ and interspecific transmission of R. felis between cofeeding arthropods on a vertebrate host. Analyses revealed that infected cat fleas transmitted R. felis to naïve cat fleas and rat fleas (Xenopsylla cheopis) via fleabite on a nonrickettsemic vertebrate host. Also, cat fleas infected by cofeeding were infectious to newly emerged uninfected cat fleas in an artificial system. Furthermore, we utilized a stochastic model to demonstrate that cofeeding is sufficient to explain the enzootic spread of R. felis amongst populations of the biological vector. Our results implicate cat fleas in the spread of R. felis amongst different vectors, and the demonstration of cofeeding transmission of R. felis through a vertebrate host represents a novel transmission paradigm for insect‐borne Rickettsia and furthers our understanding of this emerging rickettsiosis.