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

Role of Fusarium Graminearum sRNAs in Fusarium Head Blight Pathogenesis on Wheat

Fusarium graminearum causes Fusarium head blight (FHB), a devastating disease on wheat. Mycotoxin deoxynivalenol (DON) acts as a virulent factor for establishment and spread of the disease, though not required for initial infection. DON biosynthesis is highly regulated by Tri genes present on three different clusters. Small RNAs are 20-30 nucleotide non-coding RNA molecules that regulate gene expression by target cleavage, translational repression or RNA directed DNA methylation. In this study, we explored a possible role of siRNAs in DON biosynthesis pathway. When DCL2 (DICER-LIKE 2), one of the major players in sRNA biogenesis, was knocked down through Inverse Repeat Transgene method, fg-siR34 biogenesis was down regulated in the dcl2- mutant compared to the wild-type. The expression assays revealed a significant down-regulation of DON biosynthesis genes Tri4, Tri5, Tri6, Tri10 and Tri14 in the dcl2- mutant, which shows that these genes are regulated by fg-siR34. Though the growth and morphology of the dcl2- mutant remain unaffected in vivo, it was highly affected in planta since the growth of the dcl2- mutant was very less compared to the wild-type. Wheat spikes inoculated with the dcl2- mutant also showed a significant loss of DON production due to the fg-siR34 knockdown. Therefore, our data shows that DON is an important virulent factor that is necessary to F. graminearum colonization in planta. These results indicate that DON biosynthesis may be regulated by siRNAs, though the mechanism of its regulation remains to be elucidated. Furthermore, fg-milR8 a milRNA from Fusarium was also found in Fusarium infected 260-2 and 260-4. As these Near Isogenic Lines differ only for QFhb1 that is known to harbor genes for FHB resistance, this might play a role in FHB pathogenesis. mRNA targets were found only in wheat genome but not Fusarium genome. One of the targets mapped to chromosome 3B and had a similarity with XS domain of Suppressor of Gene Silencing 3 (SGS3) protein. As SGS3 proteins confer resistance to incoming pathogens (viruses) by Post Transcriptional Gene Silencing (PTGS), Fusarium might target this protein by fg-milR8 to increase pathogenesis. Mechanism of target regulation is not clear as the cleavage site could not be identified through 5’RNA Ligase mediated Rapid amplification of cDNA ends (RLM-RACE) experiments. From these results we could conclude that sRNAs play an important role in FHB pathogenesis on wheat

The Genetics and Genome-Wide Screening of Regrowth Loci, a Key Component of Perennialism in Zea diploperennis

Perennialism is common among the higher plants, yet little is known about its inheritance. Previous genetic studies of the perennialism in Zea have yielded contradictory results. In this study, we take a reductionist approach by specifically focusing on one trait: regrowth (the plant’s ability to restart a new life cycle after senescence on the same body). To address this, six hybrids were made by reciprocally crossing perennial Zea diploperennis Iltis, Doebley & R. Guzman with inbred lines B73 and Mo17 and Rhee Flint, a heirloom variety, of Z. mays L. ssp. mays. All the F1 plants demonstrated several cycles of growth, flowering, senescence and regrowth into normal flowering plants, indicating a dominant effect of the Z. diploperennis alleles. The regrowability (i.e., the plants’ ability to regrow after senescence) was stably transmitted to progeny of the hybrids. Segregation ratios of regrowth in the F2 generations are consistent with the trait controlled by two dominant, complementary loci, but do not exclude the influence of other modifiers or environment. Genome-wide screening with genotyping-by-sequencing technology indicated two major regrowth loci, regrowth 1 (reg1) and regrowth 2 (reg2), were on chromosomes 2 and 7, respectively. These findings lay the foundation for further exploration of the molecular mechanism of regrowth in Z. diploperennis. Importantly, our data indicate that there is no major barrier to transferring this trait into maize or other grass crops for perennial crop development with proper technology, which enhances sustainability of grain crop production in an environmentally friendly way