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1.
One-Enzyme RTX-PCR for the Detection of RNA Viruses from Multiple Virus Genera and Crop Plants.
Hoffmeisterová, H, Kratochvílová, K, Čeřovská, N, Slavíková, L, Dušek, J, Muller, K, Fousek, J, Plchová, H, Navrátil, O, Kundu, JK, et al
Viruses. 2022;(2)
Abstract
Reverse transcription PCR (RT-PCR) is a popular method for detecting RNA viruses in plants. RT-PCR is usually performed in a classical two-step procedure: in the first step, cDNA is synthesized by reverse transcriptase (RT), followed by PCR amplification by a thermostable polymerase in a separate tube in the second step. However, one-step kits containing multiple enzymes optimized for RT and PCR amplification in a single tube can also be used. Here, we describe an RT-PCR single-enzyme assay based on an RTX DNA polymerase that has both RT and polymerase activities. The expression plasmid pET_RTX_(exo-) was transferred to various E. coli genotypes that either compensated for codon bias (Rosetta-gami 2) or contained additional chaperones to promote solubility (BL21 (DE3) with plasmids pKJE8 or pTf2). The RTX enzyme was then purified and used for the RT-PCR assay. Several purified plant viruses (TMV, PVX, and PVY) were used to determine the efficiency of the assay compared to a commercial one-step RT-PCR kit. The RT-PCR assay with the RTX enzyme was validated for the detection of viruses from different genera using both total RNA and crude sap from infected plants. The detection endpoint of RTX-PCR for purified TMV was estimated to be approximately 0.01 pg of the whole virus per 25 µL reaction, corresponding to 6 virus particles/µL. Interestingly, the endpoint for detection of TMV from crude sap was also 0.01 pg per reaction in simulated crude plant extracts. The longest RNA fragment that could be amplified in a one-tube arrangement was 2379 bp long. The longest DNA fragment that could be amplified during a 10s extension was 6899 bp long. In total, we were able to detect 13 viruses from 11 genera using RTX-PCR. For each virus, two to three specific fragments were amplified. The RT-PCR assay using the RTX enzyme described here is a very robust, inexpensive, rapid, easy to perform, and sensitive single-enzyme assay for the detection of plant viruses.
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2.
Localization and Mechanical Transmission of Tomato Brown Rugose Fruit Virus in Tomato Seeds.
Salem, NM, Sulaiman, A, Samarah, N, Turina, M, Vallino, M
Plant disease. 2022;(1):275-281
Abstract
Tomato brown rugose fruit virus (ToBRFV), belonging to the genus Tobamovirus, is a highly virulent emerging virus, causing disease outbreaks and significant crop losses worldwide. The growing number of ToBRFV epidemic episodes prompted the investigation of the role of seeds in the dissemination of the virus as an important aspect in the overall disease management. Therefore, the objectives of this study were to determine the localization of ToBRFV within tomato seeds and to evaluate its seed transmission characteristics. Seeds extracted from naturally ToBRFV-infected tomato fruits were tested for the presence of the virus using serological, molecular, and biological assays. Three immunolocalization techniques were used to determine the localization and distribution of ToBRFV within the different tissues and parts of tomato seeds. To evaluate seed transmission of ToBRFV, two grow-out experiments were conducted to assess the rate of both vertical (seeds to progeny seedlings) and possible horizontal transmission (plant to plant) based on serological and molecular assays. Seeds extracted from ToBRFV-infected fruits had a 100% contamination rate. The localization of ToBRFV in tomato seeds is only external on the seed coat (testa). Seed transmission rate from seeds to their seedlings was very low (0.08%), while no transmission was recorded from plants to plants in a small-scale greenhouse experimental setup. In conclusion, ToBRFV is a seedborne virus located externally on tomato seed coat and transmitted mechanically from ToBRFV-contaminated tomato seeds to seedlings, which could initiate a disease foci and eventually drive further dissemination and spread of the disease in a new growing area.
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3.
Viral Reservoir Capacity of Wild Prunus Alternative Hosts of Plum Pox Virus Through Multiple Cycles of Transmission and Dormancy.
Collum, TD, Stone, AL, Sherman, DJ, Damsteegt, VD, Schneider, WL, Rogers, EE
Plant disease. 2022;(1):101-106
Abstract
Plum pox virus (PPV) is a significant pathogen of Prunus worldwide and is known for having a broad experimental host range. Many of these hosts represent epidemiological risks as potential wild viral reservoirs. A comparative study of the PPV reservoir capacity of three commonly found native North American species, western choke cherry (Prunus virginiana var. demissa), black cherry (Prunus serotina), and American plum (Prunus americana) was conducted. Pennsylvania isolates of PPV-D were transmitted from the original host peach (Prunus persica cv. GF305) to all three species. Viral accumulation and transmission rates to alternative hosts and peach were monitored over the course of five vegetative growth and cold induced dormancy (CID) cycles. The three alternative host species demonstrated differences in their ability to maintain PPV-D and the likelihood of transmission to additional alternative hosts or back transmission to peach. Western choke cherry had low (5.8%) initial infection levels, PPV-D was not transmissible to additional western choke cherry, and transmission of PPV-D from western choke cherry to peach was only possible before the first CID cycle. Black cherry had intermediate initial infection levels (26.6%) but did not maintain high infection levels after repeated CID cycles. Conversely, American plum had a high level (50%) of initial infection that was not significantly different from initial infection in peach (72.2%) and maintained moderate levels (15 to 25%) of infection and PPV-D transmission to both American plum and peach through all five cycles of CID. Our results indicate that American plum has the greatest potential to act as a reservoir host for Pennsylvania isolates of PPV-D.
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4.
Harnessing phytomicrobiome signals for phytopathogenic stress management.
Sharma, A, Raina, M, Kumar, D, Singh, A, Chugh, S, Jain, S, Kumar, M, Rustagi, A
Journal of biosciences. 2022
Abstract
Harnessing the phytomicrobiome offers a great opportunity to improve plant productivity and quality of food. In the recent past, several phytomicrobiome microbes have been explored for their potential involvement in increasing crop yield. This review strategically targets to harness the various dimensions of phytomicrobiome for biotic stress management of crop plants. The tripartite interaction involving plantmicrobiome-pathogen has been discussed. Positive interventions in this system so as to achieve disease tolerant plants has been forayed upon. The different signalling molecules sent out by interacting partners of phytomicrobiome have also been analysed. The novel concept of artificial microbial consortium in mitigation of pathogenic stress has also been touched upon. The aim of this review is to explore the hidden potential of phytomicrobiome diversity as a potent tool against phytopathogens, thereby improving crop health and productivity in a sustainable way.
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5.
Cell wall-associated effectors of plant-colonizing fungi.
Tanaka, S, Kahmann, R
Mycologia. 2021;(2):247-260
Abstract
Plant-colonizing fungi secrete a cocktail of effector proteins during colonization. After secretion, some of these effectors are delivered into plant cells to directly dampen the plant immune system or redirect host processes benefitting fungal growth. Other effectors function in the apoplastic space either as released proteins modulating the activity of plant enzymes associated with plant defense or as proteins bound to the fungal cell wall. For such fungal cell wall-bound effectors, we know particularly little about their molecular function. In this review, we describe effectors that are associated with the fungal cell wall and discuss how they contribute to colonization.
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6.
Creation and judicious application of a wheat resistance gene atlas.
Hafeez, AN, Arora, S, Ghosh, S, Gilbert, D, Bowden, RL, Wulff, BBH
Molecular plant. 2021;(7):1053-1070
Abstract
Disease-resistance (R) gene cloning in wheat (Triticum aestivum) has been accelerated by the recent surge of genomic resources, facilitated by advances in sequencing technologies and bioinformatics. However, with the challenges of population growth and climate change, it is vital not only to clone and functionally characterize a few handfuls of R genes, but also to do so at a scale that would facilitate the breeding and deployment of crops that can recognize the wide range of pathogen effectors that threaten agroecosystems. Pathogen populations are continually changing, and breeders must have tools and resources available to rapidly respond to those changes if we are to safeguard our daily bread. To meet this challenge, we propose the creation of a wheat R-gene atlas by an international community of researchers and breeders. The atlas would consist of an online directory from which sources of resistance could be identified and deployed to achieve more durable resistance to the major wheat pathogens, such as wheat rusts, blotch diseases, powdery mildew, and wheat blast. We present a costed proposal detailing how the interacting molecular components governing disease resistance could be captured from both the host and the pathogen through biparental mapping, mutational genomics, and whole-genome association genetics. We explore options for the configuration and genotyping of diversity panels of hexaploid and tetraploid wheat, as well as their wild relatives and major pathogens, and discuss how the atlas could inform a dynamic, durable approach to R-gene deployment. Set against the current magnitude of wheat yield losses worldwide, recently estimated at 21%, this endeavor presents one route for bringing R genes from the lab to the field at a considerable speed and quantity.
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7.
Nicotinamide Mononucleotide Potentiates Resistance to Biotrophic Invasion of Fungal Pathogens in Barley.
Ueda, K, Nakajima, Y, Inoue, H, Kobayashi, K, Nishiuchi, T, Kimura, M, Yaeno, T
International journal of molecular sciences. 2021;(5)
Abstract
Nicotinamide mononucleotide (NMN), a precursor of nicotinamide adenine dinucleotide (NAD), induces disease resistance to the Fusarium head blight fungus Fusarium graminearum in Arabidopsis and barley, but it is unknown at which stage of the infection it acts. Since the rate of haustorial formation of an obligate biotrophic barley powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh) was significantly reduced in NMN-treated coleoptile epidermal cells, the possibility that NMN induces resistance to the biotrophic stage of F. graminearum was investigated. The results show that NMN treatment caused the wandering of hyphal growth and suppressed the formation of appressoria-like structures. Furthermore, we developed an experimental system to monitor the early stage of infection in real-time and analyzed the infection behavior. We observed that the hyphae elongated windingly by NMN treatment. These results suggest that NMN potentiates resistance to the biotrophic invasion of F. graminearum as well as Bgh.
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8.
Plant SWEETs: from sugar transport to plant-pathogen interaction and more unexpected physiological roles.
Breia, R, Conde, A, Badim, H, Fortes, AM, Gerós, H, Granell, A
Plant physiology. 2021;(2):836-852
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Abstract
Sugars Will Eventually be Exported Transporters (SWEETs) have important roles in numerous physiological mechanisms where sugar efflux is critical, including phloem loading, nectar secretion, seed nutrient filling, among other less expected functions. They mediate low affinity and high capacity transport, and in angiosperms this family is composed by 20 paralogs on average. As SWEETs facilitate the efflux of sugars, they are highly susceptible to hijacking by pathogens, making them central players in plant-pathogen interaction. For instance, several species from the Xanthomonas genus are able to upregulate the transcription of SWEET transporters in rice (Oryza sativa), upon the secretion of transcription-activator-like effectors. Other pathogens, such as Botrytis cinerea or Erysiphe necator, are also capable of increasing SWEET expression. However, the opposite behavior has been observed in some cases, as overexpression of the tonoplast AtSWEET2 during Pythium irregulare infection restricted sugar availability to the pathogen, rendering plants more resistant. Therefore, a clear-cut role for SWEET transporters during plant-pathogen interactions has so far been difficult to define, as the metabolic signatures and their regulatory nodes, which decide the susceptibility or resistance responses, remain poorly understood. This fuels the still ongoing scientific question: what roles can SWEETs play during plant-pathogen interaction? Likewise, the roles of SWEET transporters in response to abiotic stresses are little understood. Here, in addition to their relevance in biotic stress, we also provide a small glimpse of SWEETs importance during plant abiotic stress, and briefly debate their importance in the particular case of grapevine (Vitis vinifera) due to its socioeconomic impact.
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9.
Severe Stunting Symptoms upon Nepovirus Infection Are Reminiscent of a Chronic Hypersensitive-like Response in a Perennial Woody Fruit Crop.
Martin, IR, Vigne, E, Velt, A, Hily, JM, Garcia, S, Baltenweck, R, Komar, V, Rustenholz, C, Hugueney, P, Lemaire, O, et al
Viruses. 2021;(11)
Abstract
Virus infection of plants can result in various degrees of detrimental impacts and disparate symptom types and severities. Although great strides have been made in our understanding of the virus-host interactions in herbaceous model plants, the mechanisms underlying symptom development are poorly understood in perennial fruit crops. Grapevine fanleaf virus (GFLV) causes variable symptoms in most vineyards worldwide. To better understand GFLV-grapevine interactions in relation to symptom development, field and greenhouse trials were conducted with a grapevine genotype that exhibits distinct symptoms in response to a severe and a mild strain of GFLV. After validation of the infection status of the experimental vines by high-throughput sequencing, the transcriptomic and metabolomic profiles in plants infected with the two viral strains were tested and compared by RNA-Seq and LC-MS, respectively, in the differentiating grapevine genotype. In vines infected with the severe GFLV strain, 1023 genes, among which some are implicated in the regulation of the hypersensitive-type response, were specifically deregulated, and a higher accumulation of resveratrol and phytohormones was observed. Interestingly, some experimental vines restricted the virus to the rootstock and remained symptomless. Our results suggest that GFLV induces a strain- and cultivar-specific defense reaction similar to a hypersensitive reaction. This type of defense leads to a severe stunting phenotype in some grapevines, whereas others are resistant. This work is the first evidence of a hypersensitive-like reaction in grapevine during virus infection.
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10.
Surviving the odds: From perception to survival of fungal phytopathogens under host-generated oxidative burst.
Singh, Y, Nair, AM, Verma, PK
Plant communications. 2021;(3):100142
Abstract
Fungal phytopathogens pose a serious threat to global crop production. Only a handful of strategies are available to combat these fungal infections, and the increasing incidence of fungicide resistance is making the situation worse. Hence, the molecular understanding of plant-fungus interactions remains a primary focus of plant pathology. One of the hallmarks of host-pathogen interactions is the overproduction of reactive oxygen species (ROS) as a plant defense mechanism, collectively termed the oxidative burst. In general, high accumulation of ROS restricts the growth of pathogenic organisms by causing localized cell death around the site of infection. To survive the oxidative burst and achieve successful host colonization, fungal phytopathogens employ intricate mechanisms for ROS perception, ROS neutralization, and protection from ROS-mediated damage. Together, these countermeasures maintain the physiological redox homeostasis that is essential for cell viability. In addition to intracellular antioxidant systems, phytopathogenic fungi also deploy interesting effector-mediated mechanisms for extracellular ROS modulation. This aspect of plant-pathogen interactions is significantly under-studied and provides enormous scope for future research. These adaptive responses, broadly categorized into "escape" and "exploitation" mechanisms, are poorly understood. In this review, we discuss the oxidative stress response of filamentous fungi, their perception signaling, and recent insights that provide a comprehensive understanding of the distinct survival mechanisms of fungal pathogens in response to the host-generated oxidative burst.