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Implementing effective monitoring strategies is fundamental to protecting crops from pathogens and ensuring sufficient food supply as the global population continues to grow. This is especially important for emergent plant pathogens such as tomato brown rugose fruit virus (ToBRFV), which overcomes the genetic resistance resources used in tomato breeding against tobamoviruses and has become a pandemic in less than a decade. Here, we report the development of a CRISPR/Cas12a-based test to detect ToBRFV in the laboratory and potentially in a field setting. Using different tobamoviruses to assess specificity, our test showed a clear positive signal for ToBRFV-infected samples, whereas no cross-reactivity was observed for closely related viruses. Next, we compared the limit of detection of our CRISPR-based test with a widely used reference real-time quantitative PCR test, revealing similar sensitivities for both tests. Finally, to reduce complexity and achieve field applicability, we used a fast nucleic acid purification step and compared its results side by side with those of a commonly used column-mediated protocol. The new protocol saved time and resources but at the expense of sensitivity. However, it could still be useful to confirm ToBRFV detection in samples with incipient symptoms of infection. Although there is room for improvement, to our knowledge, this is the first field-compatible CRISPR-based test to detect ToBRFV that combines isothermal amplification with a simplified nucleic acid extraction protocol. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Plant viruses can evolve towards new pathogenic entities that may eventually cause outbreaks and become epidemics or even pandemics. Seven years ago, tomato brown rugose fruit virus (ToBRFV) emerged, overcoming the genetic resistance that had been employed for more than sixty years against tobamoviruses in tomato. Since then, ToBRFV has spread worldwide, producing significant losses in tomato crops. While new resistances are deployed, the only means of control is the implementation of effective prevention and eradication strategies. For this purpose, in this work, we have designed, assessed, and compared an array of tests for the specific and sensitive detection of the ToBRFV in leaf samples. First, two monoclonal antibodies were generated against a singular peptide of the ToBRFV coat protein; antibodies were utilized to devise a double-antibody-sandwich enzyme-linked immunosorbent assay (DAS-ELISA) test that sensitively detects this virus and has no cross-reactivity with other related tobamoviruses. Second, a real-time quantitative PCR (RT-qPCR) test targeting the RNA-dependent replicase open reading frame (ORF) was designed, and its performance and specificity validated in comparison with the CaTa28 and CSP1325 tests recommended by plant protection authorities in Europe. Third, in line with the tendency to use field-deployable diagnostic techniques, we developed and tested two sets of loop-mediated isothermal amplification (LAMP) primers to double-check the detection of the movement protein ORF of ToBRFV, and one set that works as an internal control. Finally, we compared all of these methods by employing a collection of samples with different ToBRFV loads to evaluate the overall performance of each test., Work at the CEBAS-CSIC lab was partially supported by grant RTI2018-097099-B-I00 from the Spanish Ministry of Science and Innovation. Work at Abiopep was partially supported by grant PTQ-2019-010500.

Melon necrotic spot virus (MNSV) is endemic in cucurbit crops worldwide, causing epidemic outbreaks from time to time. MNSV is transmitted in nature by a soil-inhabiting fungus and also through seeds, making its detection in seed certification programs a necessity. Polyclonal antisera and RT-PCR-based detection assays have been developed for MNSV, but up to now no monoclonal antibodies (mAbs) have been described for this virus. In this study, we have produced mAbs in BALB/c mice against the MNSV over-expressed coat protein (CP). Titers of the antibodies produced against the recombinant MNSV CP ranged around 10-3 - 10-4 and the IgG yields for each mAb from ascitic fluids ranged from 1.51 to 6 mg/mL. Supernatants from ten hybridoma cell lines were evaluated in Western blot analysis and seven of them efficiently recognized the MNSV CP in crude extracts of MNSV-infected leaf material; the 2D4H4 hybridoma cell line was selected for further purification and characterization. The isotype of the 2D4H4 immunoglobulin class was identified as IgG2a and kappa light-chain. Western-blot analyses showed that mAb 2D4H4 provided sensitive and specific detection of MNSV. A TAS-ELISA protocol was developed for mAb 2D4H4. Using this protocol, limits of detection of 1:20,480 and 1:10,240 (g/mL, w/v) were attained for the homologous isolate and a heterologous MNSV isolate, respectively. Moreover, mAb 2D4H4 was used successfully to localize the MNSV CP in infected cells by immunocytochemistry/transmission electron microscopy, illustrating the usefulness of this mAb for advanced cellular studies.

We used high-throughput sequencing to identify viruses on tomato samples showing virus-like symptoms. Samples were collected from crops in the Iberian Peninsula. Either total RNA or double-stranded RNA (dsRNA) were used as starting material to build the cDNA libraries. In total, seven virus species were identified, with pepino mosaic virus being the most abundant one. The dsRNA input provided better coverage and read depth but missed one virus species compared with the total RNA input. By performing in silico analyses, we determined a minimum sequencing depth per sample of 0.2 and 1.5 million reads for dsRNA and rRNA-depleted total RNA inputs, respectively, to detect even the less abundant viruses. Primers and TaqMan probes targeting conserved regions in the viral genomes were designed and/or used for virus detection, all viruses were detected by qRT-PCR/RT-PCR in individual samples, with all except one sample showing mixed infections. Three virus species (Olive latent virus 1, Lettuce ring necrosis virus and Tomato fruit blotch virus) are herein reported for the first time in tomato crops in Spain.

The emerging lettuce big-vein disease (LBVD) is causing losses in lettuce production ranging from 30 to 70% worldwide. Several studies have associated this disease with Mirafiori lettuce big-vein virus (MiLBVV) alone or in mixed infection with lettuce big-vein associated virus (LBVaV). We used Illumina small RNA sequencing (sRNA-seq) to identify viruses present in symptomatic lettuce plants from commercial fields in Southern Spain. Data analysis using the VirusDetect tool showed the consistent presence of MiLBVV and LBVaV in diseased plants. Populations of MiLBVV and LBVaV viral small RNAs (sRNAs) were characterized, showing features essentially similar to those of other viruses, with the peculiarity of an uneven asymmetric distribution of MiLBVV virus-derived small RNAs (vsRNAs) for the different polarities of genomic RNA4 vs. RNAs1 to 3. Sanger sequencing of coat protein genes was used to study MiLBVV and LBVaV phylogenetic relationships and population genetics. The Spanish MiLBVV population was composed of isolates from three well-differentiated lineages and reflected almost all of the diversity reported for the MiLBVV species, whereas the LBVaV population showed very little genetic differentiation at the regional scale but lineage differentiation at a global geographical scale. Universal primers were used to detect and quantify the accumulation of MiLBVV and LBVaV in field samples; both symptomatic and asymptomatic plants from affected fields carried equal viral loads, with LBVaV accumulating at higher levels than MiLBVV., CG-A was recipient of grant PTQ-15-07646 from the Torres-Quevedo program (Ministry of Economy, Industry and Competitiveness; Spain) and CT of fellowship DI-14-06825 from the Industrial Doctoral program (Ministry of Economy, Industry and Competitiveness, Spain).

While recent pepino mosaic virus (PepMV; species Pepino mosaic virus, genus Potexvirus, family Alphaflexiviridae) epidemics seem to be predominantly caused by isolates of the CH2 strain, PepMV epidemics in intensive tomato crops in Spain are caused by both CH2 and EU isolates that co-circulate, representing a challenge in terms of control, including cross-protection. In this work, we hypothesized that mixed infections with two mild isolates of the EU and CH2 strains (PepMV-Sp13 and -PS5, respectively) may be useful in PepMV cross-protection in Spanish epidemics, providing protection against a broad range of aggressive isolates. Thus, we performed a range of field trials and an experimental evolution assay to determine the phenotypic and genetic stability of PepMV-Sp13 and -PS5 mixed infections, as well as their cross-protective efficiency. Our results showed that: (i) the phenotype of PepMV-Sp13 and -PS5 mixed infections was mild and did not change significantly when infecting different tomato cultivars or under different environmental conditions in Spain, (ii) PepMV-Sp13 and -PS5 mixed infections provided more efficient protection against two aggressive EU and CH2 isolates than single infections, and (iii) PepMV-Sp13 and -PS5, either in single or in mixed infections, were less variable than other two PepMV isolates occurring naturally in PepMV epidemics in Spain.

Tomato yellow leaf curl virus (TYLCV, genus Begomovirus, family Geminiviridae) is a major species that causes a tomato disease for which resistant tomato hybrids (mainly carriers of the Ty-1/Ty-3 gene) are being used widely. We have characterized begomoviruses severely affecting resistant tomato crops in Southeast Spain. Circular DNA was prepared from samples by rolling circle amplification, and sequenced by massive sequencing (2015) or cloning and Sanger sequencing (2016). Thus, 23 complete sequences were determined, all belonging to the TYLCV Israel strain (TYLCV-IL). Massive sequencing also revealed the absence of other geminiviral and beta-satellite sequences. A phylogenetic analysis showed that the Spanish isolates belonged to two groups, one related to early TYLCV-IL isolates in the area (Group 1), and another (Group 2) closely related to El Jadida (Morocco) isolates, suggesting a recent introduction. The most parsimonious evolutionary scenario suggested that the TYLCV isolates of Group 2 are back recombinant isolates derived from TYLCV-IS76, a recombinant virus currently predominating in Moroccan epidemics. Thus, an infectious Group 2 clone (TYLCV-Mu15) was constructed and used in in planta competition assays against TYLCV-IS76. TYLCV-Mu15 predominated in single infections, whereas TYLCV-IS76 did so in mixed infections, providing credibility to a scenario of co-occurrence of both types of isolates., Research in M.A.A., C.U. and M.P labs was funded by the Ministry of Economy, Industry, and Competitiveness of Spain, grant number PCIN-2017-055, and Agence Nationale de la Recherche of France, respectively, both within the ERANET-ARIMNet2 (ref. 302) program. J.A. and C.G.-A. were recipients of grants PTQ-13-05882 and PTQ-15-07646, respectively, from the Torres-Quevedo program (Ministry of Economy, Industry and Competitiveness; Spain), and C.T. of fellowship DI-14-06825 from the Industrial Doctoral program (Ministry of Economy, Industry, and Competitiveness, Spain).

The transcription initiation site of the Saccharomyces cerevisiae PRS1 gene was mapped at −179 bp. Measurement of β-galactosidase activity of the successively deleted PRS1 promoter linked to lacZ and integrated at the ura3 locus defined three DNA regions involved in the control of PRS1 expression. Gel shift analysis confirmed the data.

Six open reading frames (ORFs) of unknown function from Saccharomyces cerevisiae chromosome XV, three from the left and three from the right arm, were deleted in two diploid strains by the short flanking homology method (Wach et al., 1994). Transformants were selected as Geneticin (G418)-resistant colonies and correct integration of the kanMX4 cassette was checked by colony PCR. Following sporulation of the diploids, tetrads were dissected and scored for the segregation of the G418-resistant marker. We have developed a widely applicable method for the construction of gap repair plasmids to obtain the cognate clones for each of the disrupted ORFs. The 5'- and 3'-flanks of the ORF in question are linked by a unique restriction endonuclease. When the plasmid is cut at this site it can be used to obtain, by selection for the appropriate antibiotic resistance, long flanking homology (LFH) cassettes containing the cognate clone or the disrupted allele. The LFH cassette containing the cognate clone or the disrupted allele can be released from the gap-repaired plasmid by cutting at the inserted flanking restriction sites. One of the six ORFs (YOR319w) corresponds to an essential gene whose product is part of the spliceosome complex. Haploid as well as homozygous and heterozygous diploid disruptant strains for each of the five non-essential ORFs were subjected to growth test on different media at 15 degrees C, 30 degrees C and 37 degrees C. Disruption of YOR322c causes osmotically sensitive growth on YEPD at 37 degrees C and the product of YOL091w appears to play a role in sporulation since the homozygous diploid disruptant has lost the ability to sporulate.

In a shotgun approach we sequenced the cosmid pEOA284 containing a fragment derived from the right arm of chromosome XV of Saccharomyces cerevisiae. An analysis of the sequence revealed that it contained open reading frames (ORFs) corresponding to the known genes SLY41, SPS4, COT1, FAA1, PMT3, PRO2 and MYO2. Of the 18 unknown ORFs, five are contained totally within, and two, O6105 and O6163, partially overlap other ORFs. ORF O6116 and O6139 have putative introns. Regions of similarity with chromosomes I and XIII have been uncovered. Interestingly, most of the paired ORFs encode proteins of the same gene family. The relatedness of these ORFs suggests gene duplication.

Phosphoribosyl-pyrophosphate synthetase (Prs) catalyses the synthesis of phosphoribosyl pyrophosphate (PRPP), an intermediate in nucleotide metabolism and the biosynthesis of the amino acids histidine and tryptophan. The Saccharomyces cerevisiae genome contains a family of five PRS genes, PRS1-PRS5. Using anti-peptide antisera directed against two different epitopes of Prs1p it was shown that Prs1p localizes to granular cytoplasmic structures. This localization was confirmed by living cell microscopy of strains expressing a functional green fluorescent protein (GFP)-tagged Prs1p. Analysis of Prs1p distribution in conditional secretory-deficient (sec) mutants suggested that the observed distribution of Prs1p is independent of the secretory pathway. Electron microscopy revealed that plasma membrane invaginations and accumulation of cytoplasmic vesicles were more frequent in strains which lack some of the PRS genes than in the wild-type. The fact that Deltaprs1 and Deltaprs3 are hypersensitive to caffeine and unable to recover from exposure to it as judged by the release of alkaline phosphatase points to a possible link between Prs and the maintenance of cell integrity.

The PRS gene family in Saccharomyces cerevisiae consists of five genes each capable of encoding a 5-phosphoribosyl-1(alpha)-pyrophosphate synthetase polypeptide. To gain insight into the functional organization of this gene family we have constructed a collection of strains containing all possible combinations of disruptions in the five PRS genes. Phenotypically these deletant strains can be classified into three groups: (i) a lethal phenotype that corresponds to strains containing a double disruption in PRS2 and PRS4 in combination with a disruption in either PRS1 or PRS3; simultaneous deletion of PRS1 and PRS5 or PRS3 and PRS5 are also lethal combinations; (ii) a second phenotype that is encountered in strains containing disruptions in PRS1 and PRS3 together or in combination with any of the other PRS genes manifests itself as a reduction in growth rate, enzyme activity, and nucleotide content; (iii) a third phenotype that corresponds to strains that, although affected in their phosphoribosyl pyrophosphate-synthesizing ability, are unimpaired for growth and have nucleotide profiles virtually the same as the wild type. Deletions of PRS2, PRS4, and PRS5 or combinations thereof cause this phenotype. These results suggest that the polypeptides encoded by the members of the PRS gene family may be organized into two functional entities. Evidence that these polypeptides interact with each other in vivo was obtained using the yeast two-hybrid system. Specifically PRS1 and PRS3 polypeptides interact strongly with each other, and there are significant interactions between the PRS5 polypeptide and either the PRS2 or PRS4 polypeptides. These data suggest that yeast phosphoribosyl pyrophosphate synthetase exists in vivo as multimeric complex(es).

Summary The nucleotide sequence (6138 bp) of a microaerobically inducible region (hupV/VI) from the Rhizobium leguminosarum bv. viciae hydrogenase gene cluster has been determined. Six genes, arranged as a single operon, were identified, and designated hypA, B, F, C, D and E based on the sequence similarities of all of them, except hypF, to genes from the hydrogenase pleiotropic operon (hyp) from Escherichia coli. The gene products from hypBFCDE were identified by in vivo expression analysis in E. coli, and their molecular sizes were consistent with those predicted from the nucleotide sequence. Transposon Tn5 insertions into hypB, hypF, hypD and hypE resulted in R. leguminosarum mutants that lacked any hydrogenase activity in symbiosis with peas, but still were able to synthesize the polypeptide for the hydrogenase large subunit. The gene products HypA, HypB, HypF and HypD contained CX2C motifs characteristic of metal-binding proteins. In addition, HypB bore a long histidine-rich stretch of amino acids near the N-terminus, suggesting a possible role in nickel binding for this protein. The gene product HypF, which was translationally coupled to HypB, presented two cysteine motifs (CX2CX81CX2C) with a capacity to form zinc finger-like structures in the N-terminal third of the protein. A role in nickel metabolism in relation to hydrogenase synthesis is postulated for proteins HypB and HypF.