Grapevine (Vitis vinifera), one of the most economically important fruit crops in the world, suffers significant yield losses from powdery mildew, a major fungal disease caused by Erysiphe necator. In addition to suppressing host immunity, phytopathogens modulate host proteins termed susceptibility (S) factors to promote their proliferation in plants. In this study, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) technology was used to enable the targeted mutagenesis of MLO (mildew resistance Locus O) family genes that are thought to serve as S factors for powdery mildew fungi. Small deletions or insertions were induced in one or both alleles of two grapevine MLO genes, VvMLO3 and VvMLO4, in the transgenic plantlets of the powdery mildew-susceptible cultivar Thompson Seedless. The editing efficiency achieved with different CRISPR/Cas9 constructs varied from 0 to 38.5%. Among the 20 VvMLO3/4-edited lines obtained, one was homozygous for a single mutation, three harbored biallelic mutations, seven were heterozygous for the mutations, and nine were chimeric, as indicated by the presence of more than two mutated alleles in each line. Six of the 20 VvMLO3/4-edited grapevine lines showed normal growth, while the remaining lines exhibited senescence-like chlorosis and necrosis. Importantly, four VvMLO3-edited lines showed enhanced resistance to powdery mildew, which was associated with host cell death, cell wall apposition (CWA) and H2O2 accumulation. Taken together, our results demonstrate that CRISPR/Cas9 genome-editing technology can be successfully used to induce targeted mutations in genes of interest to improve traits of economic importance, such as disease resistance in grapevines.

/pdf/crispr-cas9-mediated-mutagenesis-of-vvmlo3-results-in-4eieupva90.pdf

CRISPR/Cas9-mediated mutagenesis of VvMLO3 results in enhanced resistance to powdery mildew in grapevine (Vitis vinifera).

During the last years, a great effort has been dedicated at the development and employment of diverse approaches for achieving more stress-tolerant and climate-flexible crops and sustainable yield increases to meet the food and energy demands of the future. The ongoing climate change is in fact leading to more frequent extreme events with a negative impact on food production, such as increased temperatures, drought, soil salinization as well as invasive arthropod pests and diseases. In this review, diverse "green strategies" (e.g., chemical priming, root-associated microorganisms), and advanced technologies (e.g., genome editing, high-throughput phenotyping) are described on the basis of the most recent research evidence. Particularly, attention has been focused on the potential use in a context of sustainable and climate-smart agriculture (the so called "next agriculture generation") to improve plant tolerance and resilience to abiotic and biotic stresses. In addition, the gap between the results obtained in controlled experiments and those from application of these technologies in real field conditions (lab to field step) is also discussed. This article is protected by copyright. All rights reserved.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/ppl.13547

New approaches to improve crop tolerance to biotic and abiotic stresses.

Traditional breeding or genetically modified organisms (GMOs) have for a long time been the sole approaches to effectively cope with biotic and abiotic stresses and implement the quality traits of crops. However, emerging diseases as well as unpredictable climate changes affecting agriculture over the entire globe force scientists to find alternative solutions required to quickly overcome seasonal crises. In this review, we first focus on cisgenesis and genome editing as challenging biotechnological approaches for breeding crops more tolerant to biotic and abiotic stresses. In addition, we take into consideration a toolbox of new techniques based on applications of RNA interference and epigenome modifications, which can be adopted for improving plant resilience. Recent advances in these biotechnological applications are mainly reported for non-model plants and woody crops in particular. Indeed, the characterization of RNAi machinery in plants is fundamental to transform available information into biologically or biotechnologically applicable knowledge. Finally, here we discuss how these innovative and environmentally friendly techniques combined with traditional breeding can sustain a modern agriculture and be of potential contribution to climate change mitigation.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/pbi.13605

Novel and emerging biotechnological crop protection approaches

A detailed landscape of CRISPR-Cas-mediated plant disease and pest management.

Climate change poses a serious threat to global agricultural activity and food production. Plant genome editing technologies have been widely used to develop crop varieties with superior qualities or can tolerate adverse environmental conditions. Unlike conventional breeding techniques (e.g., selective breeding and mutation breeding), modern genome editing tools offer more targeted and specific alterations of the plant genome and could significantly speed up the progress of developing crops with desired traits, such as higher yield and/or stronger resilience to the changing environment. In this review, we discuss the current development and future applications of genome editing technologies in mitigating the impacts of biotic and abiotic stresses on agriculture. We focus specifically on the CRISPR/Cas system, which has been the center of attention in the last few years as a revolutionary genome-editing tool in various species. We also conducted a bibliographic analysis on CRISPR-related papers published from 2012 to 2021 (10 years) to identify trends and potential in the CRISPR/Cas-related plant research. In addition, this review article outlines the current shortcomings and challenges of employing genome editing technologies in agriculture with notes on future prospective. We believe combining conventional and more innovative technologies in agriculture would be the key to optimizing crop improvement beyond the limitations of traditional agricultural practices.

https://www.mdpi.com/2223-7747/11/19/2625/pdf?version=1665467179

Genome Editing for Sustainable Crop Improvement and Mitigation of Biotic and Abiotic Stresses

Plant diseases are among the major causes of crop yield losses around the world. To confer disease resistance, conventional breeding relies on the deployment of single resistance (R) genes. However, this strategy has been easily overcome by constantly evolving pathogens. Disabling susceptibility (S) genes is a promising alternative to R genes in breeding programs, as it usually offers durable and broad-spectrum disease resistance. In Arabidopsis, the S gene DMR6 (AtDMR6) encodes an enzyme identified as a susceptibility factor to bacterial and oomycete pathogens. Here, we present a model-to-crop translational work in which we characterize two AtDMR6 orthologs in tomato, SlDMR6-1 and SlDMR6-2. We show that SlDMR6-1, but not SlDMR6-2, is up-regulated by pathogen infection. In agreement, Sldmr6-1 mutants display enhanced resistance against different classes of pathogens, such as bacteria, oomycete, and fungi. Notably, disease resistance correlates with increased salicylic acid (SA) levels and transcriptional activation of immune responses. Furthermore, we demonstrate that SlDMR6-1 and SlDMR6-2 display SA-5 hydroxylase activity, thus contributing to the elucidation of the enzymatic function of DMR6. We then propose that SlDMR6 duplication in tomato resulted in subsequent subfunctionalization, in which SlDMR6-2 specialized in balancing SA levels in flowers/fruits, while SlDMR6-1 conserved the ability to fine-tune SA levels during pathogen infection of the plant vegetative tissues. Overall, this work not only corroborates a mechanism underlying SA homeostasis in plants, but also presents a promising strategy for engineering broad-spectrum and durable disease resistance in crops.

Yu Geng

Papers

Loss of function of a DMR6 ortholog in tomato confers broad-spectrum disease resistance.