Microalgae, due to their complex metabolic capacity, are being continuously explored for nutraceuticals, pharmaceuticals, and other industrially important bioactives. However, suboptimal yield and productivity of the bioactive of interest in local and robust wild-type strains are of perennial concerns for their industrial applications. To overcome such limitations, strain improvement through genetic engineering could play a decisive role. Though the advanced tools for genetic engineering have emerged at a greater pace, they still remain underused for microalgae as compared to other microorganisms. Pertaining to this, we reviewed the progress made so far in the development of molecular tools and techniques, and their deployment for microalgae strain improvement through genetic engineering. The recent availability of genome sequences and other omics datasets form diverse microalgae species have remarkable potential to guide strategic momentum in microalgae strain improvement program. This review focuses on the recent and significant improvements in the omics resources, mutant libraries, and high throughput screening methodologies helpful to augment research in the model and non-model microalgae. Authors have also summarized the case studies on genetically engineered microalgae and highlight the opportunities and challenges that are emerging from the current progress in the application of genome-editing to facilitate microalgal strain improvement. Toward the end, the regulatory and biosafety issues in the use of genetically engineered microalgae in commercial applications are described.

Genome-editing, a recent technological advancement in the field of life sciences, is one of the great examples of techniques used to explore the understanding of the biological phenomenon. Besides having different site-directed nucleases for genome editing over a decade ago, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) based genome editing approach has become a choice of technique due to its simplicity, ease of access, cost, and flexibility. In the present review, several CRISPR/Cas based approaches have been discussed, considering recent advances and challenges to implicate those in the crop improvement programs. Successful examples where CRISPR/Cas approach has been used to improve the biotic and abiotic stress tolerance, and traits related to yield and plant architecture have been discussed. The review highlights the challenges to implement the genome editing in polyploid crop plants like wheat, canola, and sugarcane. Challenges for plants difficult to transform and germline-specific gene expression have been discussed. We have also discussed the notable progress with multi-target editing approaches based on polycistronic tRNA processing, Csy4 endoribonuclease, intron processing, and Drosha ribonuclease. Potential to edit multiple targets simultaneously makes it possible to take up more challenging tasks required to engineer desired crop plants. Similarly, advances like precision gene editing, promoter bashing, and methylome-editing will also be discussed. The present review also provides a catalog of available computational tools and servers facilitating designing of guide-RNA targets, construct designs, and data analysis. The information provided here will be useful for the efficient exploration of technological advances in genome editing field for the crop improvement programs.

Rice is a global food grain crop for more than one-third of the human population and a source for food and nutritional security. Rice production is subjected to various stresses; blast disease caused by Magnaporthe oryzae is one of the major biotic stresses that has the potential to destroy total crop under severe conditions. In the present review, we discuss the importance of rice and blast disease in the present and future global context, genomics and molecular biology of blast pathogen and rice, and the molecular interplay between rice–M. oryzae interaction governed by different gene interaction models. We also elaborated in detail on M. oryzae effector and Avr genes, and the role of noncoding RNAs in disease development. Further, rice blast resistance QTLs; resistance (R) genes; and alleles identified, cloned, and characterized are discussed. We also discuss the utilization of QTLs and R genes for blast resistance through conventional breeding and transgenic approaches. Finally, we review the demonstrated examples and potential applications of the latest genome-editing tools in understanding and managing blast disease in rice.

Leaf rust is one of the most important diseases of wheat and is caused by Puccinia triticina, a highly variable rust pathogen prevalent worldwide. Decoding the genome of this pathogen will help in unraveling the molecular basis of its evolution and in the identification of genes responsible for its various biological functions. We generated high quality draft genome sequences (approximately 100- 106 Mb) of two races of P. triticina; the variable and virulent Race77 and the old, avirulent Race106. The genomes of races 77 and 106 had 33X and 27X coverage, respectively. We predicted 27678 and 26384 genes, with average lengths of 1,129 and 1,086 bases in races 77 and 106, respectively and found that the genomes consisted of 37.49% and 39.99% repetitive sequences. Genome wide comparative analysis revealed that Race77 differs substantially from Race106 with regard to segmental duplication (SD), repeat element, and SNP/InDel characteristics. Comparative analyses showed that Race 77 is a recent, highly variable and adapted Race compared with Race106. Further sequence analyses of 13 additional pathotypes of Race77 clearly differentiated the recent, active and virulent, from the older pathotypes. Average densities of 2.4 SNPs and 0.32 InDels per kb were obtained for all P. triticina pathotypes. Secretome analysis demonstrated that Race77 has more virulence factors than Race 106, which may be responsible for the greater degree of adaptation of this pathogen. We also found that genes under greater selection pressure were conserved in the genomes of both races, and may affect functions crucial for the higher levels of virulence factors in Race77. This study provides insights into the genome structure, genome organization, molecular basis of variation, and pathogenicity of P. triticina. The genome sequence data generated in this study have been submitted to public domain databases and will be an important resource for comparative genomics studies of the more than 4000 existing Puccinia species.

Magnaporthe oryzae infection causes rice blast, a destructive disease that is responsible for considerable decrease in rice yield. Development of resistant varieties via introgressing resistance genes with marker-assisted breeding can eliminate pesticide use and minimize crop losses. Here, resistant near-isogenic line (NIL) of Pusa Basmati-1(PB1) carrying broad spectrum rice blast resistance gene Pi9 was used to investigate Pi9-mediated resistance response. Infected and uninfected resistant NIL and susceptible control line were subjected to RNA-Seq. With the exception of one gene (Pi9), transcriptional signatures between the two lines were alike, reflecting basal similarities in their profiles. Resistant and susceptible lines possessed 1043 (727 up-regulated and 316 down-regulated) and 568 (341 up-regulated and 227 down-regulated) unique and significant differentially expressed loci (SDEL), respectively. Pathway analysis revealed higher transcriptional activation of kinases, WRKY, MYB and ERF transcription factors, JA-ET hormones, chitinases, glycosyl hydrolases, lipid biosynthesis, pathogenesis and secondary metabolism related genes in resistant NIL than susceptible line. Singular enrichment analysis demonstrated that blast resistant NIL is significantly enriched with genes for primary and secondary metabolism, response to biotic stimulus and transcriptional regulation. The co-expression network showed proteins of genes in response to biotic stimulus interacted in a manner unique to resistant NIL upon M. oryzae infection. These data suggest that Pi9 modulates genome-wide transcriptional regulation in resistant NIL but not in susceptible PB1. We successfully used transcriptome profiling to understand the molecular basis of Pi9-mediated resistance mechanisms, identified potential candidate genes involved in early pathogen response and revealed the sophisticated transcriptional reprogramming during rice-M. oryzae interactions.