SpRY greatly expands the genome editing scope in rice with highly flexible PAM recognition.

BACKGROUND: Plant genome engineering mediated by various CRISPR-based tools requires specific protospacer adjacent motifs (PAMs), such as the well-performed NGG, NG, and NNG, to initiate target recognition, which notably restricts the editable range of the plant genome. RESULTS: In this study, we thoroughly investigate the nuclease activity and the PAM preference of two structurally engineered SpCas9 variants, SpG and SpRY, in transgenic rice. Our study shows that SpG nuclease favors NGD PAMs, albeit less efficiently than the previously described SpCas9-NG, and that SpRY nuclease achieves efficient editing across a wide range of genomic loci, exhibiting a preference of NGD as well as NAN PAMs. Furthermore, SpRY-fused cytidine deaminase hAID*Δ and adenosine deaminase TadA8e are generated, respectively. These constructs efficiently induce C-to-T and A-to-G conversions in the target genes toward various non-canonical PAMs, including non-G PAMs. Remarkably, high-frequency self-editing events (indels and DNA fragments deletion) in the integrated T-DNA fragments as a result of the nuclease activity of SpRY are observed, whereas the self-editing of SpRY nickase-mediated base editor is quite low in transgenic rice lines. CONCLUSIONS: The broad PAM compatibility of SpRY greatly expands the targeting scope of CRISPR-based tools in plant genome engineering.

Sixty Years After the First Description: Genome Sequence and Biological Characterization of European Wheat Striate Mosaic Virus Infecting Cereal Crops.

High-throughput sequencing technologies were used to identify plant viruses in cereal samples surveyed from 2012 to 2017. Fifteen genome sequences of a tenuivirus infecting wheat, oats, and spelt in Estonia, Norway, and Sweden were identified and characterized by their distances to other tenuivirus sequences. Like most tenuiviruses, the genome of this tenuivirus contains four genomic segments. The isolates found from different countries shared at least 92% nucleotide sequence identity at the genome level. The planthopper Javesella pellucida was identified as a vector of the virus. Laboratory transmission tests using this vector indicated that wheat, oats, barley, rye, and triticale, but none of the tested pasture grass species (Alopecurus pratensis, Dactylis glomerata, Festuca rubra, Lolium multiflorum, Phleum pratense, and Poa pratensis), are susceptible. Taking into account the vector and host range data, the tenuivirus we have found most probably represents European wheat striate mosaic virus first identified about 60 years ago. Interestingly, whereas we were not able to infect any of the tested cereal species mechanically, Nicotiana benthamiana was infected via mechanical inoculation in laboratory conditions, displaying symptoms of yellow spots and vein clearing evolving into necrosis, eventually leading to plant death. Surprisingly, one of the virus genome segments (RNA2) encoding both a putative host systemic movement enhancer protein and a putative vector transmission factor was not detected in N. benthamiana after several passages even though systemic infection was observed, raising fundamental questions about the role of this segment in the systemic spread in several hosts.

A Novel Recombined Potato virus Y Isolate in China.

This study reports the findings of a distinct Potato virus Y (PVY) isolate found in Northeast China. One hundred and ten samples (leaves and tubers) were collected from potato plants showing mosaic symptoms around the city of Harbin in Heilongjiang province of China. The collected tubers were planted and let to grow in a greenhouse. New potato plants generated from these tubers showed similar symptoms, except for one plant. Subsequent serological analyses revealed PVY as the causing agent of the disease. A novel PVY isolate (referred to as HLJ-C-44 in this study) was isolated from this sample showing unique mild mosaic and crisped leaf margin symptoms. The complete genome of this isolate was analyzed and determined. The results showed that HLJ-C-44 is a typical PVY isolate. Phylogenetic analysis indicated that this isolate belongs to the N-Wi strain group of PVY recombinants (PVYN-Wi) and also shared the highest overall sequence identity (nucleotide and amino acid) with other members of this strain group. However, recombination analysis of isolate HLJ-C-44 revealed a recombination pattern that differed from that of other PVYN-Wi isolates. Moreover, biological assays in four different potato cultivars and in Nicotiana tabacum also revealed a different phenotypic response than that of a typical PVYN-Wi isolate. This data, combined, suggest that HLJ-C-44 is a novel PVY recombinant with distinct biological properties.

Symptomless infectious cDNA clone of a Norwegian isolate of Poinsettia mosaic virus.

An infectious cDNA clone of a Norwegian isolate of Poinsettia mosaic virus (PnMV) was generated. It consisted of 6,098 nucleotides and encoded a polyprotein of 219.5 kDa. Sequence comparisons indicated that this isolate shared 98.6% (nucleotide) and 97.1% (amino acid) identity with the previously sequenced isolate from Germany. RNA transcripts derived from this cDNA were infectious in Nicotiana benthamiana. However, plants did not present typical PnMV symptoms. Furthermore, RNA transcripts from this cDNA clone were not infectious in poinsettia. Serial propagation of this cDNA clone in N. benthamiana plants restored symptom induction in this host but did not re-establish infectivity in poinsettia.

Potyviral 6K2 protein long-distance movement and symptom-induction functions are independent and host-specific.

Deletion of various portions, or insertion of six histidine residues (6xHis) into various positions of the membrane-bound 6K2 protein (53 amino acids) of Potato virus A (PVA, genus Potyvirus), inhibited systemic infection in Nicotiana tabacum and N. benthamiana plants. However, a spontaneous mutation (Gly2Cys) that occurred in 6K2 adjacent to the 6xHis insert placed between Ser1 and Gly2 enabled systemic infection in a single N. benthamiana plant. No symptoms were observed, but virus titers were similar to the symptom-inducing wild-type (wt) PVA. N. tabacum plants were not systemically infected, albeit virus propagation was observed in inoculated protoplasts. The 6xHis/Gly2Cys mutant was reconstructed in vitro and serially propagated by mechanical inoculation in N. benthamiana. Following the third passage, a novel viral mutant appeared, lacking the last four His residues of the insert, as well as the Gly2 and Thr3 of 6K2. It infected N. tabacum plants systemically, and in the systemically infected N. benthamiana leaves, vein chlorosis and mild yellowing symptoms were observed, typical of wt PVA infection. The mutant virus accumulated to titers similar to wt PVA in both hosts. These results show that the PVA 6K2 protein affects viral long-distance movement and symptom induction independently and in a host-specific manner.

Two potato proteins, including a novel RING finger protein (HIP1), interact with the potyviral multifunctional protein HCpro.

Potyviral helper-component proteinase (HCpro) is a multifunctional protein exerting its cellular functions in interaction with putative host proteins. In this study, cellular protein partners of the HCpro encoded by Potato virus A (PVA) (genus Potyvirus) were screened in a potato leaf cDNA library using a yeast two-hybrid system. Two cellular proteins were obtained that interact specifically with PVA HCpro in yeast and in the two in vitro binding assays used. Both proteins are encoded by single-copy genes in the potato genome. Analysis of the deduced amino acid sequences revealed that one (HIP1) of the two HCpro interactors is a novel RING finger protein. The sequence of the other protein (HIP2) showed no resemblance to the protein sequences available from databanks and has known biological functions.