CRISPR-Cas9 Targeting of the eIF4E1 Gene Extends the Potato Virus Y Resistance Spectrum of the Solanum tuberosum L. cv. Desirée.

Translation initiation factors and, in particular, the eIF4E family are the primary source of recessive resistance to potyviruses in many plant species. However, no eIF4E-mediated resistance to this virus genus has been identified in potato (Solanum tuberosum L.) germplasm. As in tomato, the potato eIF4E gene family consists of eIF4E1, its paralog eIF4E2, eIF(iso)4E, and nCBP. In tomato, eIF4E1 knockout (KO) confers resistance to a subset of potyviruses, while the eIF4E1/2 double KO, although conferring a broader spectrum of resistance, leads to plant developmental defects. Here, the tetraploid potato cv. Desirée owning the dominant Ny gene conferring resistance to potato virus Y (PVY) strain O but not NTN was used to evaluate the possibility to expand its PVY resistance spectrum by CRISPR-Cas9-mediated KO of the eIF4E1 susceptibility gene. After a double process of plant protoplast transfection-regeneration, eIF4E1 KO potatoes were obtained. The knockout was specific for the eIF4E1, and no mutations were identified in its eIF4E2 paralog. Expression analysis of the eIF4E family shows that the disruption of the eIF4E1 does not alter the RNA steady-state level of the other family members. The eIF4E1 KO lines challenged with a PVYNTN isolate showed a reduced viral accumulation and amelioration of virus-induced symptoms suggesting that the eIF4E1 gene was required but not essential for its multiplication. Our data show that eIF4E1 editing can be usefully exploited to broaden the PVY resistance spectrum of elite potato cultivars, such as Desirée, by pyramiding eIF4E-mediated recessive resistance.

Distinct Effects of p19 RNA Silencing Suppressor on Small RNA Mediated Pathways in Plants.

RNA silencing is one of the main defense mechanisms employed by plants to fight viruses. In change, viruses have evolved silencing suppressor proteins to neutralize antiviral silencing. Since the endogenous and antiviral functions of RNA silencing pathway rely on common components, it was suggested that viral suppressors interfere with endogenous silencing pathway contributing to viral symptom development. In this work, we aimed to understand the effects of the tombusviral p19 suppressor on endogenous and antiviral silencing during genuine virus infection. We showed that ectopically expressed p19 sequesters endogenous small RNAs (sRNAs) in the absence, but not in the presence of virus infection. Our presented data question the generalized model in which the sequestration of endogenous sRNAs by the viral suppressor contributes to the viral symptom development. We further showed that p19 preferentially binds the perfectly paired ds-viral small interfering RNAs (vsiRNAs) but does not select based on their sequence or the type of the 5' nucleotide. Finally, co-immunoprecipitation of sRNAs with AGO1 or AGO2 from virus-infected plants revealed that p19 specifically impairs vsiRNA loading into AGO1 but not AGO2. Our findings, coupled with the fact that p19-expressing wild type Cymbidium ringspot virus (CymRSV) overcomes the Nicotiana benthamiana silencing based defense killing the host, suggest that AGO1 is the main effector of antiviral silencing in this host-virus combination.

Transcriptomics of tomato plants infected with TYLCSV or expressing the central TYLCSV Rep protein domain uncover changes impacting pathogen response and senescence.

To establish a successful infection viruses need to overcome plant innate immune responses and redirect host gene expression for their multiplication and diffusion. Tomato yellow leaf curl Sardinia virus (TYLCSV) is a geminivirus, which causes significant economic losses in tomato. The multifunctional replication associated geminivirus protein (Rep) has an important role during viral infection. In particular, the Rep central domain spanning from aa 120 to 180 is known to interact with viral and host factors. In this study, we used long serial analysis of gene expression to analyse the transcriptional profiles of transgenic tomato plants expressing the first 210 amino acids of TYLCSV Rep (Rep210) and TYLCSV-infected wild-type tomato plants (Wt-Ty). Also, we compared these profiles with those of transgenic Rep130 tomatoes. Comparison of Wt-Ty and Rep210 libraries with the wild-type one identified 118 and 203 differentially expressed genes (DEGs), respectively. Importantly, 55% of Wt-Ty DEGs were in common with Rep210, and no ones showed opposite expression. Conversely, a negligible overlap was found between Rep130 DEGs and Wt-Ty and Rep210 ones. TYLCSV- and Rep210-repressed genes, but not induced ones, overlapped with the leaf senescence process. Interestingly, TYLCSV upregulates expression of genes involved in the negative regulation of programmed cell death (PCD), several of which were also regulated by the abscisic acid. Rep210 upregulated genes related to defence response, immune system processes and negative regulation of PCD. Collectively, our results support a model in which the Rep central domain has a pivotal role in redirecting host plant gene expression.

Robust RNA silencing-mediated resistance to Plum pox virus under variable abiotic and biotic conditions.

Some abiotic and biotic conditions are known to have a negative impact on post-transcriptional gene silencing (PTGS), thus representing a potential concern for the production of stable engineered virus resistance traits. However, depending on the strategy followed to achieve PTGS of the transgene, different responses to external conditions can be expected. In the present study, we utilized the Nicotiana benthamiana­Plum pox virus (PPV) pathosystem to evaluate in detail the stability of intron-hairpin(ihp)-mediated virus resistance under conditions known to adversely affect PTGS. The ihp plants grown at low or high temperatures were fully resistant to multiple PPV challenges, different PPV inoculum concentrations and even to a PPV isolate differing from the ihp construct by more than 28% at the nucleotide level. In addition, infections of ihp plants with viruses belonging to Cucumovirus, Potyvirus or Tombusvirus, all known to affect PTGS at different steps, were not able to defeat PPV resistance. Low temperatures did not affect the accumulation of transgenic small interfering RNAs (siRNAs), whereas a clear increase in the amount of siRNAs was observed during infections sustained by Cucumber mosaic virus and Potato virus Y. Our results show that the above stress factors do not represent an important concern for the production,through ihp-PTGS technology, of transgenic plants having robust virus resistance traits.

Tomato yellow leaf curl Sardinia virus-resistant tomato plants expressing the multifunctional N-terminal domain of the replication-associated protein show transcriptional changes resembling stress-related responses.

The N-terminal domain (amino acids 1-130) of the replication-associated protein (Rep130 ) of Tomato yellow leaf curl Sardinia virus (TYLCSV) retains the ability of full-length Rep to localize to the nucleus and to down-regulate C1 transcription when ectopically expressed in plants, both functions being required to inhibit homologous viral replication. In this study, we analysed the effect of Rep130 expression on virus resistance and the plant transcriptome in the natural and agronomically important host species of TYLCSV, Solanum lycopersicum. Tomato plants accumulating high levels of Rep130 were generated and proved to be resistant to TYLCSV. Using an in vitro assay, we showed that plant-expressed Rep130 also retains the catalytic activity of Rep, thus supporting the notion that this protein domain is fully functional. Interestingly, Rep130 -expressing tomatoes were characterized by an altered transcriptional profile resembling stress-related responses. Notably, the serine-type protease inhibitor (Ser-PI) category was over-represented among the 20 up-regulated genes. The involvement of Rep130 in the alteration of host mRNA steady-state levels was confirmed using a distinct set of virus-resistant transgenic tomato plants expressing the same TYLCSV Rep130 , but from a different, synthetic, gene. Eight genes were found to be up-regulated in both types of transgenic tomato and two encoded Ser-PIs. Four of these eight genes were also up-regulated in TYLCSV-infected wild-type tomato plants. Implications with regard to the ability of this Rep domain to interfere with viral infections and to alter the host transcriptome are discussed.

From immunity to susceptibility: virus resistance induced in tomato by a silenced transgene is lost as TGS overcomes PTGS.

Tomato line 30.4 was obtained engineering the nucleocapsid (N) gene of tomato spotted wilt virus into plant genome, and immunity to tomato spotted wilt virus infection of its self-pollinated homozygous progeny was observed. Despite the presence of a high amount of transgenic transcripts, transgenic proteins have not been detected, suggesting a mechanism of resistance mediated by RNA. In the present study, we identify post-transcriptional gene silencing as the main mechanism of resistance, which is able to spread systemically through grafting, and show that the line 30.4 resistant plants produce both 24 and 21-22 nt N-gene specific siRNA classes. The transgenic locus in chromosome 4 shows complex multiple insertions of four T-DNA copies in various orientations, all with 3' end deletions in the terminator and part of the N gene. However, for three of them, polyadenylated transcripts are produced, due to flanking tomato genome sequences acting as alternative terminators. Interestingly, starting at the fifth generation after the transformation event, some individual plants show a tomato spotted wilt virus-susceptible phenotype. The change is associated with the disappearance of transgene-specific transcripts and siRNAs, and with hyper-methylation of the transgene, which proceeds gradually through the generations. Once it reaches a critical threshold, the shift from post-transcriptional gene silencing to transcriptional silencing of the transgene eliminates the previously well established virus resistance.

An RGG sequence in the replication-associated protein (Rep) of Tomato yellow leaf curl Sardinia virus is involved in transcriptional repression and severely impacts resistance in Rep-expressing plants.

Truncated versions of the replication-associated protein (Rep) of Tomato yellow leaf curl Sardinia virus (TYLCSV) can interfere with various viral functions and the N-terminal 130 aa are sufficient for strongly inhibiting C1-gene transcription and virus replication and confer resistance in transgenic plants. In this work, we analysed the relevance of an RGG sequence at aa 124-126, highly conserved in begomoviruses, in these inhibitory functions as well as in the subcellular localization of Rep. Although no role of this RGG sequence was detected by cell fractionation and immunogold labelling in Rep localization, this sequence appears relevant for the transcriptional control of the C1-gene and for the inhibition of viral replication and dramatically impacts resistance in transgenic plants. These results are discussed in the context of the model of Rep-mediated resistance against TYLCSV.

Tomato yellow leaf curl Sardinia virus can overcome transgene-mediated RNA silencing of two essential viral genes.

To evaluate RNA silencing for the control of geminivirus infection, two classes of post-transcriptionally silenced (PTS) plants were tested using Tomato yellow leaf curl Sardinia virus (TYLCSV) Rep-210-transgenic plants, a sensexantisense hybrid and two multicopy sense lines. In both classes, PTS plants accumulated low or undetectable amounts of Rep-210 protein and mRNA but high amounts of Rep-210 small interfering RNAs. PTS plants were susceptible to TYLCSV when challenged by agroinoculation or using high viruliferous whitefly (Bemisia tabaci) pressure, although some plants were resistant at low whitefly pressure. Delayed infections were also observed, indicating that TYLCSV could overcome transgene silencing of rep and of the nested C4 gene. TYLCSV infection boosted transgene silencing but this did not lead to recovery. The data suggest that if the virus reaches a threshold level of expression/replication in the initially infected cells then virus spreading can no longer be prevented.

Tomato yellow leaf curl Sardinia virus rep-derived resistance to homologous and heterologous geminiviruses occurs by different mechanisms and is overcome if virus-mediated transgene silencing is activated.

The replication-associated protein (Rep) of geminiviruses is involved in several biological processes brought about by the presence of distinct functional domains. Recently, we have exploited the multifunctional character of the Tomato yellow leaf curl Sardinia virus (TYLCSV) Rep to develop a molecular interference strategy to impair TYLCSV infection. We showed that transgenic expression of its N-terminal 210 amino acids (Rep-210) confers resistance to the homologous virus by inhibiting viral transcription and replication. We have now used biochemical and transgenic approaches to carry out a fuller investigation of the molecular resistance mechanisms in transgenic plants expressing Rep-210. We show that Rep-210 confers resistance through two distinct molecular mechanisms, depending on the challenging virus. Resistance to the homologous virus is achieved by the ability of Rep-210 to tightly inhibit C1 gene transcription, while that to heterologous virus is due to the interacting property of the Rep-210 oligomerization domain. Furthermore, we present evidence that in Rep-210-expressing plants, the duration of resistance is related to the ability of the challenging virus to shut off transgene expression by a posttranscriptional homology-dependent gene silencing mechanism. A model of Rep-210-mediated geminivirus resistance that takes transgene- and virus-mediated mechanisms into account is proposed.

A viral protein suppresses RNA silencing and binds silencing-generated, 21- to 25-nucleotide double-stranded RNAs.

Posttranscriptional gene silencing (PTGS) processes double-stranded (ds) RNAs into 21-25 nucleotide (nt) RNA fragments that direct ribonucleases to target cognate mRNAs. In higher plants, PTGS also generates mobile signals conferring sequence-specific silencing in distant organs. Since PTGS acts as an antiviral system in plants, successful virus infection requires evasion or suppression of gene silencing. Here we report that the 19 kDa protein (p19) of tombusviruses is a potent silencing suppressor that prevents the spread of mobile silencing signal. In vitro, p19 binds PTGS-generated, 21-25 nt dsRNAs and 21-nt synthetic dsRNAs with 2-nt 3' overhanging end(s), while it barely interacts with single-stranded (ss) RNAs, long dsRNAs or blunt-ended 21-nt dsRNAs. We propose that p19 mediates silencing suppression by sequestering the PTGS-generated 21-25 nt dsRNAs, thus depleting the specificity determinants of PTGS effector complexes. Moreover, the observation that p19-expressing transgenic plants show altered leaf morphology might indicate that the p19-targeted PTGS pathway is also important in the regulation of plant development.