File Name: resistance from bacterial and fungal diseases in plants .zip
This is the sixth fact sheet in a series of ten designed to provide an overview of key concepts in plant pathology.
Plants play different important roles in the environment such as ecosystem balance and food supplement for animals and humans. Moreover, wild or cultivated plants are considered the powerful biofertilizers for the soil, where the plant debris after death and degradation provides the soil with sufficient organic matters. Accordingly, plant care is a great duty and hard mission, which must be constantly improved. The study of plant pathogens belongs to the branch of biology known as plant pathology.
Genome editing technologies have progressed rapidly and become one of the most important genetic tools in the implementation of pathogen resistance in plants. As regards the achievement of viral disease resistance, the main strategies employed in model species such as Arabidopsis and Nicotiana benthamiana , which include the integration of CRISPR-encoding sequences that target and interfere with the viral genome and the induction of a CRISPR-mediated targeted mutation in the host plant genome, will be discussed.
After spending years deciphering and reading genomes, researchers are now editing and rewriting them to develop crop plants resistant to specific pests and pathogens. Plant breeding has been the most successful approach for developing new crop varieties since domestication occurred, making possible major advances in feeding the world and societal development.
Crops are susceptible to a large set of pathogens including fungi, bacteria, and viruses, which cause important economic losses FAO, ; the enhancement of plant resistance plays an important role in adjusting crop production to meet global population increases. Approaches to disease control that depend on resistant varieties and agrochemicals are usually highly effective whenever they are deployed. However, due to the high evolutionary potential of many plant pathogens, novel genotypes no longer sensitive to the resistance gene or the phytosanitary product can rapidly emerge via mutation or recombination.
When this happens, particular disease control approaches can rapidly be rendered ineffective as the novel genotypes increase in frequency through natural selection and quickly spread to other locations, causing failure of control over large geographic areas.
An understanding of interactions between plants and communities of bacteria, fungi, and other microorganisms has been a major area of investigation for many years. The advent of high-throughput molecular technologies has made a more complete inventory of the pathogens associated with particular crops possible, and provided insight into how these communities may be affected by environmental factors and the crop genotype. During pre-genomic years, traditional breeding programs were based on the identification of natural and induced mutant alleles for resistance, and their incorporation into elite genotypes through breeding techniques.
These approaches were uncertain and imprecise, leading for instance to the transfer of large genome regions instead of just single gene insertions. Nevertheless, mutation breeding methods have been quite successful in improving disease resistance, and traditional plant breeding has been used to generate new crop varieties for decades.
Numerous mutants have been developed through mutation induction, showing enhanced resistance to various diseases. Among the most widely known mutants are those induced at the mildew resistance locus MLO in barley for resistance to powdery mildew Miklis et al.
The mlo mutant is interesting, as the allele has not broken down and has provided unprecedented resistance to mildew for two decades Panstruga and Schulze-Lefert, This longevity is due to a gene knockout.
In other cases where resistance to specific pathotypes is conferred by a specific host gene allele, mutagenesis needs to be deployed to provide more precise single nucleotide mutations in the target gene sequence. The revolution driven by the availability of genome and transcriptome sequences offers a new start for plant breeding programs. Association genetics based on single nucleotide polymorphisms SNPs and other molecular markers are spreading in plant breeding, creating high throughput data fundamental for the identification of quantitative trait loci QTL.
Major QTL are employed in crops to provide quantitative resistance to pathogens, together with the use of major resistance R genes introduced into varieties with superior agronomic characteristics. New breeding techniques NBTs are attracting attention in plant research and concern many different areas, such as developmental biology, abiotic stress tolerance or plant-pathogen resistance Nelson et al.
NBT include the most recent and powerful molecular approaches for precise genetic modifications of single or multiple gene targets. They employ site-directed nucleases to introduce double stranded breaks at predetermined sites in DNA.
These breaks are repaired by different host cell repair mechanisms, resulting either in small insertions or deletions via near homologous end-joining NHEJ or micro-homology-mediated end-joining MMEJ , or in a modified gene carrying predetermined nucleotide changes copied from a repair matrix via homologous recombination HR. The requirement of a protospacer adjacent motif PAM limits the possible target sequences in a gene of interest. This limitation is of minor importance if the aim is simply to inactivate a gene by targeted mutagenesis at any position.
It has much more importance for genome editing aiming at the precise change of specific nucleotides in a gene. Consequently, major efforts are under way to find Cas9-like proteins with different PAM sequences or to engineer the original Cas9 from S. Cpf1 is also considerably smaller than Cas9, is capable of RNAse activity to process its guide RNA, and introduces a staggered double break, which can be useful for enhancing homology-directed recombination and generating efficient gene insertion.
Illustrative diagram of Cas9 and Cpf1 activities. The Cas9 is colored in light blue and the Cpf1 in dark blue; in black is represented the gRNA for gene targeting. B Gene target activity. C DNA ends after nuclease activity. Cas9 lead to blunt-end and Cpf1 to sticky-ends. The ease of multiplexing, i. For example, the simultaneous mutation of 14 different genes by a single construct has been demonstrated in Arabidopsis Peterson et al.
In crops, several multiplex genome editing MGE strategies were reported early on Ma et al. Constructs alternating sgRNA and tRNA sequences under the control of a single U3 or U6 promoter permit reduction of the size of the construct and limit the risk of silencing due to direct repetitions of promoter sequences.
Starting from a previous study on gene silencing of TaLpx-1 , which encodes the wheat 9-lipoxygenase resistance gene to Fusarium graminearum Nalam et al.
The PTG system containing gRNA activity was validated in wheat confirming gene editing efficacy and providing an effective tool for rapid trait pyramiding in breeding programs. A PTG primary transcript. Two types of off-target effects are evoked by scientists and regulatory agencies: i expected off-target in genome regions with high sequence similarity to the target and ii unexpected off-target in unrelated genome regions.
The former is generally addressed by PCR amplification and sequencing of regions known to be similar to the target, the latter by whole genome sequencing Feng et al. Genome sequence information is necessary for the prediction of expected off-target effects. The PAM functions as a recognition site outside of the targeted element and does not give specificity for nuclease cleavage Shah et al. Such state-of-the art design is effortless if the gene is unique in the genome, but it becomes rather challenging if the gene has one or more paralogs.
This also means that the design is generally easier for diploid genomes without recent duplications than for recently duplicated or polyploid genomes.
In silico genome analysis of potential target sequences in dicots and monocots has confirmed that, as expected, larger genomes contain more PAMs and more potential targets Bortesi et al. High specificity of between Analysis of expected off-target sites, with only one to several mismatches with the primary target, has revealed that the position of the mismatches in the sequence is significant.
Unwanted off-target mutations become more frequent when mismatches are located far from the seed region Zhang et al. The results were different for the three genes: highly conserved for TaGW2 target sequence was specific for all three genomes , moderate for TaLpx-1 target sequence specific in two genomes , and low for TaMLO. The study reveals the discrepancy in gRNA prediction and mutagenesis efficiency, confirming that gRNA with low predicted efficiency can achieve high mutation frequency even though the prediction suggested different targets with high mutagenesis scores.
Empirical testing seems necessary in order to avoid putative gRNA inefficiency. Unexpected off-target mutations do not occur at a frequency above the spontaneous mutation rate of plants. Private companies seem to prefer biolistic transformation to Agrobacterium -mediated transformation in the case of gene editing with donor template Shi et al. Even though both transformation processes have decent efficiencies nowadays, they remain limited to the above genotypes with poor agronomic traits.
This limitation has recently been overcome by the overexpression of Baby boom Bbm and Wuschel2 Wus2 genes, which stimulated callus growth and increased the overall transformation frequency in maize, including in recalcitrant genotypes. Proof of concept has also been provided for enhanced transformation in sorghum Lowe et al.
In rice, most genotypes can easily be transformed both via Agrobacterium -mediated transformation and by biolistic methods. As for maize, but involving a higher number of studies, protoplast transient assay is becoming an efficient tool for testing CRISPR-target before starting the transformation of embryos or scutellum derived calli by Agrobacterium or particle bombardment Gao et al. Regeneration of rice protoplasts is still very challenging, but important optimization efforts may render it feasible in the near future.
Higher numbers of mutants were reported with the Agrobacterium -mediated compared to the biolistic transformation approach. In conclusion, although preferences for certain delivery methods exist for certain species, efficiency is not only linked to the technique itself, but also to the know-how of a given lab as regards a given technique.
The importance of preliminary screens will certainly increase with the foreseeable shift from targeted mutagenesis to repair matrix based genome editing, which will increase the number of events to analyze due to lower efficiency. RNP technology has been established in plants and may help toward exemption from regulatory oversight, but its efficiency needs to be improved to make it a routine tool.
Advances in genome editing tools have opened new ways to achieve the improvement of resistance in crops. Plant viruses are a serious threat to many economically important staple and specialty crops. Geminiviridae is a large family of plant viruses causing worldwide crop losses among several important families, such as Cucurbitaceae, Euphorbiaceae, Solanaceae, Malvaceae, and Fabaceae Zaidi et al. The virus genome is replicated through a rolling-circle amplification mechanism via a dsDNA replicative form, or by recombination-mediated replication Hanley-Bowdoin et al.
The most important genus of geminiviruses in economic terms is Begomovirus. Ji et al. Similar findings were described by Baltes et al. Ali et al. All sgRNAs were able to interfere with TYLCV genome sequences, but targeting the stem-loop invariant sequence contained in the IR caused a more significant reduction of viral replication and accumulation. The results showed attenuated symptoms for both viruses, demonstrating that mixed infection immunity can be developed via a single sgRNA specific for conserved sequences of multiple viral strains.
Furthermore, Ali et al. Conversely, no novel variants were detected in N. Even though the NHEJ machinery repaired the double strand breaks caused by the Cas9 protein, the IR-repaired variants generated virus genomes unable to replicate, thus providing a better overall interference with the viral life cycle. However, the search for and characterization of related nucleases has led to the discovery of enzymes that can bind to and cut RNA, such as FnCas9 from Francisella novicida or LwaCas13a from Leptotrichia wadei.
A first report demonstrating resistance to RNA viruses Zhang et al. Furthermore, the resistance obtained by expressing the sgRNA-FnCas9 system was quite stable and still active in the T6 generation.
Importantly, Zhang et al. Moreover, in contrast with the previously described interference with geminivirus replication in the nucleus, no nuclear localization signal is necessary for FnCas9, which interferes with the RNA viruses in the cytoplasm.
Similar work has been carried out with Cas13a. Aman et al. Four different viral genomic regions were targeted: two targets in the green fluorescent protein GFP region, one in the helper component proteinase silencing suppressor HC-Pro , and one in the coat protein CP. All the systems aiming at protection against viruses described so far require the maintenance of a transgene for Cas9 and sgRNA in the genome of the crop plants, rendering them subject to genetically modified organism GMO regulation.
Chandrasekaran et al. Conversely, heterozygous knockout plants and non-mutant plants did not reveal any resistance to these viruses. A similar editing approach was utilized by Pyott et al. Furthermore, homozygous T3 eIF iso 4E mutants did not significantly differ in growth and development compared to wild-type plants. Recently, Macovei et al. The RTSV-resistant T 2 plants obtained did not show any detectable mutation in the off-target sites and were negative when tested for the presence of Cas9.
Furthermore, after inoculation with RTSV, agronomic parameters such as plant height and grain yield were enhanced in the edited rice plants compared to their wild-type counterparts under glasshouse conditions.
The advantage of knocking out host susceptibility genes is that this is a relatively simple method that renders following the mutation easy. The loss of a host factor required for the viral life cycle is a form of recessive resistance that should be more durable than that of dominant R genes because viruses undergo a lower selective pressure, preventing their evolution to hinder host defense mechanisms. A possible disadvantage of the knockout strategy is that it may negatively influence plant vigor, supporting the selection of virus variants breaking the resistance, as already observed in nature Abdul-Razzak et al.
PLoS Pathog 15 6 : e This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Pesticide use, breeding of resistance genes, and genetic manipulation of plant immune components have helped to mitigate this threat. However, rapid evolution of pathogen resistance and virulence, together with host range expansion and host jumps, contribute to severe disease outbreaks, especially in the context of current agricultural practices [ 2 ].
These diseases are caused by conditions external to the plant, not living agents. They cannot spread from plant to plant, but are very common and should be considered when assessing the health of any plant. Examples of abiotic diseases include nutritional deficiencies, soil compaction, salt injury, ice, and sun scorch Figure These diseases are caused by living organisms. They are called plant pathogens when they infect plants.
This page provides an overview of the fungal diseases in vegetable crops. The related tools listed at the end of the page provided detailed information about identification, symptoms, and management of fungal diseases. It is important to have a plant diagnostics laboratory confirm the pathogen causing any diseases in a crop so that the disease can be appropriately managed. Fungi constitute the largest number of plant pathogens and are responsible for a range of serious plant diseases.
Genome editing technologies have progressed rapidly and become one of the most important genetic tools in the implementation of pathogen resistance in plants. As regards the achievement of viral disease resistance, the main strategies employed in model species such as Arabidopsis and Nicotiana benthamiana , which include the integration of CRISPR-encoding sequences that target and interfere with the viral genome and the induction of a CRISPR-mediated targeted mutation in the host plant genome, will be discussed. After spending years deciphering and reading genomes, researchers are now editing and rewriting them to develop crop plants resistant to specific pests and pathogens. Plant breeding has been the most successful approach for developing new crop varieties since domestication occurred, making possible major advances in feeding the world and societal development. Crops are susceptible to a large set of pathogens including fungi, bacteria, and viruses, which cause important economic losses FAO, ; the enhancement of plant resistance plays an important role in adjusting crop production to meet global population increases.
Medical illustration of Candida spp. Antifungal drugs treat fungal infections by killing or stopping the growth of dangerous fungi in the body. Fungi, like bacteria, can develop antibiotic resistance , when germs like bacteria and fungi develop the ability to defeat the drugs designed to kill them.
Густые клубы пара окутывали корпус ТРАНСТЕКСТА, ступеньки лестницы были влажными от конденсации, она едва не упала, поскользнувшись. Она нервничала, гадая, сколько еще времени продержится ТРАНСТЕКСТ. Сирены продолжали завывать; то и дело вспыхивали сигнальные огни. Тремя этажами ниже дрожали и гудели резервные генераторы. Сьюзан знала, что где-то на дне этого погруженного в туман подземелья есть рубильник.
На каждом из них красовалась печать АНБ. - Хочешь посмотреть, чем занимаются люди в шифровалке? - спросил он, заметно нервничая. - Вовсе нет, - ответила Мидж. - Хотела бы, но шифровалка недоступна взору Большого Брата. Ни звука, ни картинки. Приказ Стратмора. Все, что я могу, - это проверить статистику, посмотреть, чем загружен ТРАНСТЕКСТ.
Он хотел прикоснуться к ней, но не посмел.
Беккер слушал как завороженный. Учитель превратился в ученика. Однажды вечером на университетском представлении Щелкунчика Сьюзан предложила Дэвиду вскрыть шифр, который можно было отнести к числу базовых. Весь антракт он просидел с ручкой в руке, ломая голову над посланием из одиннадцати букв: HL FKZC VD LDS В конце концов, когда уже гасли огни перед началом второго акта, его осенило.
Вы немец. Мужчина нерешительно кивнул. Беккер заговорил на чистейшем немецком: - Мне нужно с вами поговорить. Мужчина смотрел на него недовольно. - Was wollen Sie.
Смотрите внимательно, - предупредил Смит. - Халохот - профессионал. Это его первый выстрел в публичном месте.
Ш-ш-ш, - утешал он. - Это. Теперь все в порядке. Сьюзан не могла унять дрожь. - Ком… мандер, - задыхаясь, пробормотала она, сбитая с толку.
И улыбнулся, едва сохраняя спокойствие.
- Чем же отличаются эти чертовы изотопы. Никто этого не знает? - Ответа он не дождался. Техники и все прочие беспомощно смотрели на ВР. Джабба повернулся к монитору и вскинул руки.
Понятия не имею. - Похож на китайца. Японец, подумал Беккер. - Бедняга.
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