In-planta Gene Targeting in Barley Using Cas9 With and Without Geminiviral Replicons

Advances in the use of RNA-guided Cas9-based genome editing in plants have been rapid over the last few years. A desirable application of genome editing is gene targeting (GT), as it allows a wide range of precise modifications; however, this remains inefficient especially in key crop species. Here, we describe successful, heritable gene targeting in barley at the target site of Cas9 using an in-planta strategy but fail to achieve the same using a wheat dwarf virus replicon to increase the copy number of the repair template. Without the replicon, we were able to delete 150 bp of the coding sequence of our target gene whilst simultaneously fusing in-frame mCherry in its place. Starting from 14 original transgenic plants, two plants appeared to have the required gene targeting event. From one of these T0 plants, three independent gene targeting events were identified, two of which were heritable. When the replicon was included, 39 T0 plants were produced and shown to have high copy numbers of the repair template. However, none of the 17 lines screened in T1 gave rise to significant or heritable gene targeting events despite screening twice the number of plants in T1 compared with the non-replicon strategy. Investigation indicated that high copy numbers of repair template created by the replicon approach cause false-positive PCR results which are indistinguishable at the sequence level to true GT events in junction PCR screens widely used in GT studies. In the successful non-replicon approach, heritable gene targeting events were obtained in T1, and subsequently, the T-DNA was found to be linked to the targeted locus. Thus, physical proximity of target and donor sites may be a factor in successful gene targeting.

A real-time loop-mediated isothermal amplification for detection of the wheat dwarf virus in wheat and the insect vector Psammotettix alienus

Wheat dwarf virus (WDV, genus Mastrevirus, family Geminiviridae) is an economically important and widespread pathogen of cereal crops. It causes huge yield loss in wheat due to the unavailability of resistant varieties and rapid transmission by the vector leafhopper, Psammotettix alienus (Dahlb). To monitor and forecast this viral disease, an early diagnosis method is required for WDV detection in both infected plants and the virus vectors. In this study, we developed a real-time loop-mediated isothermal amplification (LAMP) assay for WDV detection. The positive sample could be detected within 28-32 min by following a simple and cost-effective procedure. The real-time LAMP assay showed a sensitivity of 2.7×105-6 copies/µL for detection and a high specificity for WDV amplification, with a similar accuracy to qPCR. Furthermore, a tube-closed dye method facilitates the inspection of the LAMP reaction and avoids cross contamination in the detection of the virus. This valuable detection assay could serve as an important tool for diagnosis and forecasting wheat dwarf disease intensity in the field.

Wheat dwarf virus and its impact on the 2020 harvest in some regions of Ukraine

Goal. Identify the virus that causes symptoms of dwarfism and non-earing of winter wheat (reduced or no ear formation), and investigate its effect on plant yields. Methods. Visual diagnostics, enzyme-linked immunosorbent assay in double sandwich modification (DAS-ELISA), polymerase chain reaction, biometric, determination of yield and its structure, statistical data processing. Results. A study of winter wheat varieties from Vinnytsia, Khmelnytsky, Kyiv, Chernihiv and Cherkasy regions with symptoms of dwarfism, yellowing of leaves and non-earing. ELISA and PCR have shown that the disease is caused by Wheat dwarf virus. The absence of Wheat streak mosaic virus, Barley yellow dwarf virus, Brome mosaic virus and Wheat spindle mosaic virus (Wheat streak mosaic virus) was shown in the studied samples. It was found that the dwarf wheat virus significantly reduces the number of grains in the ear (3.3 times), the weight of grains from the ear (3.4 times) and the weight of 1000 grains (1.9—3.3 times) depending on the degree of damage plants (from moderate to severely affected). Conclusions. The circulation of dwarf wheat virus in agrocenoses of five regions of Ukraine and a significant negative impact of the disease on the yield of winter wheat plants have been established. The obtained results indicate the need for constant monitoring and testing of plants for the presence of wheat dwarf virus in Ukraine.

Development of loop-mediated isothermal amplification assay for rapid detection of genetically different wheat dwarf virus isolates

Wheat dwarf virus (WDV) is considered as one of the most common viruses on cereal crops. Recently, severe outbreaks of WDV have been observed especially on winter wheat in southwestern part of Poland. Moreover, the presence of genetically different WDV-barley-specific and WDV-wheat-specific forms (WDV-B and WDV-W, respectively) was confirmed. In this study, a loop-mediated isothermal amplification assay (LAMP) was developed for the first time for efficient and rapid detection of WDV-B and WDV-W in infected plants. The reaction was performed using a set of three primer pairs: WDVF3/WDVB3, WDVFIB/WDVBIP and WDVLoopF/WDVLoopB specific for coat protein coding sequence. The amplified products were analyzed by direct staining of DNA, gel electrophoresis and real-time monitoring of the amplification curves. The sensitivity of optimized reaction was tenfold higher in comparison with conventional PCR. LAMP assay developed here is a useful and practical method for the rapid detection of different WDV isolates and can be implemented by phytosanitary services.

Incidence and Distribution of Insect-Transmitted Cereal Viruses in Wheat in China from 2007 to 2019

Diseases caused by insect-transmitted viruses are the predominant constraint to wheat production worldwide. However, detailed knowledge of virus incidence and dynamics in China in recent years is very limited. Here, major wheat-growing regions of China were surveyed over 10 years for insect-transmitted viruses, and 2,143 samples were collected (in 2007 to 2015) and analyzed by molecular hybridization or multiplex reverse-transcription PCR for barley yellow dwarf viruses (BYDVs: BYDV-GAV, -GPV, and -PAV) and wheat dwarf virus (WDV). In a 4-year survey (2016 to 2019), the incidence of eight insect-transmitted viruses (BYDVs, WDV, wheat yellow striate virus [WYSV], barley yellow striate mosaic virus [BYSMV], northern cereal mosaic virus [NCMV], and rice black-streaked dwarf virus [RBSDV]) was investigated, and BYDVs and WDV were widely distributed across China. BYDV-GAV (29.0% of the tested sample) was the most abundant, followed by BYDV-PAV (23.2%) from 2007 to 2015. From 2016 to 2019, however, BYDV-PAV had become the predominant species (39.5% positive of 952 samples tested), while the incidence of BYDV-GAV (13.4%) had declined. During the entire survey, the incidence of BYDV-GPV was very low in some locations in northwestern and northern China, and all eight viruses caused only local epidemics, not large-scale outbreaks throughout China. Two new cereal-infecting rhabdoviruses, leafhopper-transmitted WYSV and planthopper-transmitted BYSMV, were also found in China in recent years.