The CRISPR-Cas system: its origin, functions and applications in biotechnology: A Review

The CRISPR-Cas system is a genome editing system seen in prokaryotic immune system. Bacteria and archaea protect itself against invading viruses and plasmid by targeting RNA or DNA of the invading element predominantly using this gene-editing tool. The CRISPR- Cas defense mechanism is carried out in three stages; adaptation stage where the spacers are inserted into the CRISPR locus, the expression stage where crRNA is formed by transcription of the CRISPR loci and the interference stage where the invading element is destroyed by the crRNA and cas proteins. The CRISPR-cas has been involved in many other functions apart from the immune defense they include; DNA repair, regulation of virulence, genome evolution, inhibit biofilm formation etc. The application of CRISPR-Cas system include genome engineering, agriculture to efficiently target and mutate plants, improve crop yield and crop resistance, in medicine to eradicate genetic diseases. However, ethical considerations are a major setback of CRISPR-Cas application especially in medicine. CRISPR-Cas has been used in variety of species including cultured human cell, rice, drosophila and mice.

Genetic Analysis Reveals Three Novel QTLs Underpinning a Butterfly Egg-Induced Hypersensitive Response-Like Cell Death in Brassica Rapa

Background Cabbage white butterflies (Pieris spp.) can be severe pests of Brassica crops such as Chinese cabbage, Pak choi (Brassica rapa) or cabbages (B. oleracea). Eggs of Pieris spp. can induce a hypersensitive response-like (HR-like) cell death which reduces egg survival in the wild black mustard (B. nigra). Unravelling the genetic basis of this egg-killing trait in Brassica crops could improve crop resistance to herbivory, reducing major crop losses and pesticides use. Here we investigated the genetic architecture of a HR-like cell death induced by P. brassicae eggs in B. rapa. Results A germplasm screening of B. rapa 56 accessions, representing the genetic and geographical diversity of a B. rapa core collection, showed phenotypic variation for cell death. An image-based phenotyping protocol was developed to accurately measure size of HR-like cell death and was then used to identify two accessions that consistently showed weak (R-o-18) or strong cell death response (L58). Screening of 160 RILs derived from these two accessions resulted in three novel QTLs for Pieris brassicae-induced cell death on chromosomes A02 (Pbc1), A03 (Pbc2), and A06 (Pbc3). The three QTLs Pbc1-3 contain cell surface receptors, intracellular receptors and other genes involved in plant immunity processes, such as ROS accumulation and cell death formation. Synteny analysis with A. thaliana suggested that Pbc1 and Pbc2 are novel QTLs associated with this trait, while Pbc3 contains also LecRK-I.1, a gene of A. thaliana previously associated with cell death induced by a P. brassicae egg extract. Conclusions This study provides the first genomic regions associated with the Pieris egg-induced HR-like cell death in a Brassica crop species. It is a step closer towards unravelling the genetic basis of an egg-killing crop resistance trait, paving the way for breeders to further fine-map and validate candidate genes.

A laboratory method for mass rearing the orange wheat blossom midge, Sitodiplosis mosellana (Diptera: Cecidomyiidae)

Orange wheat blossom midge, Sitodiplosis mosellana (Géhin) (Diptera: Cecidomyiidae), has been successfully reared in the laboratory for more than 20 years in Winnipeg, Manitoba, Canada. The rearing method has been developed to the point where it efficiently produces large numbers of wheat midge continuously under laboratory conditions for use in experiments on wheat midge biology and for screening wheat lines for crop resistance. Adult survival was extended by providing high humidity, and oviposition was increased by simulating natural dawn and dusk conditions and by supplying preflowering spring wheat to adults. Preventing desiccation of the wheat midge larvae in the wheat spikes before overwintering in soil and providing optimal cold conditions for a long enough period to break larval diapause enabled successful adult emergence. We provide data to facilitate the coordination of timing of wheat midge emergence from diapause with the wheat susceptible period. The method can be readily scaled up for screening many lines for resistance or scaled down for small experiments. Here, we report details of the rearing method so that others can implement it for research on the management of this internationally important pest.

Different Host Plants Distinctly Influence the Feeding Ability of the Brown Citrus Aphid Toxoptera citricida

Piercing–sucking insects are important crop pests, and an understanding of their feeding behavior and population development plays a crucial role in studying insect population dynamics and crop resistance. In our study, we examined the probing behavior of the brown citrus aphid, Toxoptera citricida, using electropenetrography and assessed its population development after 8 days on seven host plants: Yuzu, Citrus junos Sieb. ex Tanaka; Rough Lemon, C. jambhiri Lush.; ‘Luofu’ kumquat, Fortunella margarita Swingle; ‘Olinda’ valencia orange, C. sinensis (L.) Osbeck; ‘Yanxiwanlu’ Ponkan, C. reticulata Blanco; ‘Rohde Red’ valencia orange, C. sinensis; and ‘Eureka’ lemon, C. limon (L.) Osbeck. The results demonstrated that probing by the brown citrus aphid differed significantly according to the target hosts. Toxoptera citricida produced significantly more pathway activities on Eureka than on Rough Lemon and Yuzu. Toxoptera citricida spent more time from the first probe to first salivation into phloem sieve elements on Eureka compared to Yuzu. In addition, the total duration of ingestion from sieve cells of each aphid in the phloem-feeding phase was shortest on Eureka, and this was significantly shorter than that on Yuzu, Rough Lemon, Luofu, and Olinda. The population number of T. citricida on Eureka after 8 days was significantly lower than that on the other hosts. Overall, Eureka was found to have obvious resistance to T. citricida, whereas Yuzu and Rough Lemon were susceptible host plants. These results provide a theoretical basis for exploring aphid-resistant fruit tree resources using resistant varieties.

Fusarium oxysporum f. sp. niveum Molecular Diagnostics Past, Present and Future

Watermelon is an important commercial crop in the Southeastern United States and around the world. However, production is significantly limited by biotic factors including fusarium wilt caused by the hemibiotrophic fungus Fusarium oxysporum forma specialis niveum (Fon). Unfortunately, this disease has increased significantly in its presence over the last several decades as races have emerged which can overcome the available commercial resistance. Management strategies include rotation, improved crop resistance, and chemical control, but early and accurate diagnostics are required for appropriate management. Accurate diagnostics require molecular and genomic strategies due to the near identical genomic sequences of the various races. Bioassays exist for evaluating both the pathogenicity and virulence of an isolate but are limited by the time and resources required. Molecular strategies are still imperfect but greatly reduce the time to complete the diagnosis. This article presents the current state of the research surrounding races, both how races have been detected and diagnosed in the past and future prospects for improving the system of differentiation. Additionally, the available Fon genomes were analyzed using a strategy previously described in separate formae speciales avirulence gene association studies in Fusarium oxysporum races.

Analysis of wild plant pathogen populations reveals a signal of adaptation in genes evolving for survival in agriculture in the beet rust pathogen (Uromyces beticola)

Improvements in crop resistance to pathogens can reduce yield losses and address global malnourishment today. Gene-for-gene -type interactions can identify new sources of resistance but genetic resistance is often short lived. Ultimately an understanding of how pathogens rapidly adapt will allow us to both increase resistance gene durability and more effectively target chemical treatments. Until recently all agricultural pathogens were living on wild hosts. To understand crop pathogen evolution, we compared genetic diversity in agricultural and wild populations. Wild reservoirs may be the source of emergent pathogen lineages, but here we outline a strategy for comparison of wild and agricultural pathogen populations to highlight genes adapting to agriculture. To address this, we have selected and developed the beet rust system (Beta vulgaris, Uromyces beticola, respectively) as our wild-agricultural model. Our hypothesis is that pathogen adaptation to agricultural crops will be evident as divergence in comparisons of wild and agricultural plant pathogen populations. We sampled isolates in both the wild and agriculture, sequenced and assembled and annotated a large fungal genome and analysed genetic diversity in 42 re-sequenced rust isolates. We found population differentiation between isolates in the wild compared to a predominantly agricultural group. Fungal effector genes are co-evolving with host resistance and are important for successful colonisation. We predicted (and found) that these exhibit a greater signal of diversification and adaptation and more importantly displayed increased wild agricultural divergence. Finding a signal of adaptation in these genes highlights this as an important strategy to identify genes which are key to pathogen success, that analysis of agricultural isolates alone cannot.

Parasitic Plants in Agriculture and Management

Parasitic plants are among the most problematic pests of agricultural crops worldwide. They are found worldwide in all plant communities except aquatic. Parasitic plants are the organisms that settle in the host plant by means of the special organs they have developed and penetrate the vascular tissues of the hosts and meet their nutritional, water and mineral needs from the host plant. This particular body they have is called a haustorium. The discovery and investigation of the haustorium structures led to the evaluation of many heterotrophic plant species previously defined as parasitic plants in different groups. Host organisms are very important in completing the life cycle of parasitic plants. In general, the parasite weakens the host, so it produces fewer flowers and viable seeds or the value of the timber is reduced. However, some parasites, mostly annual root parasites belonging to the Orobanchaceae, can kill the host and cause significant economic damage while attacking monocultures in agriculture, and much effort is put into controlling these harmful parasites. Parasitic weeds are difficult to control because there are few resources for crop resistance and it is difficult to apply sufficiently selective control methods to kill weeds without physically and biochemically damaging the crop to which they are attached.

Spotlight on the Roles of Whitefly Effectors in Insect–Plant Interactions

The Bemisia tabaci species complex (whitefly) causes enormous agricultural losses. These phloem-feeding insects induce feeding damage and transmit a wide range of dangerous plant viruses. Whiteflies colonize a broad range of plant species that appear to be poorly defended against these insects. Substantial research has begun to unravel how phloem feeders modulate plant processes, such as defense pathways, and the central roles of effector proteins, which are deposited into the plant along with the saliva during feeding. Here, we review the current literature on whitefly effectors in light of what is known about the effectors of phloem-feeding insects in general. Further analysis of these effectors may improve our understanding of how these insects establish compatible interactions with plants, whereas the subsequent identification of plant defense processes could lead to improved crop resistance to insects. We focus on the core concepts that define the effectors of phloem-feeding insects, such as the criteria used to identify candidate effectors in sequence-mining pipelines and screens used to analyze the potential roles of these effectors and their targets in planta. We discuss aspects of whitefly effector research that require further exploration, including where effectors localize when injected into plant tissues, whether the effectors target plant processes beyond defense pathways, and the properties of effectors in other insect excretions such as honeydew. Finally, we provide an overview of open issues and how they might be addressed.