In silico analysis of fungal small RNA accumulation reveals putative plant mRNA targets in the symbiosis between an arbuscular mycorrhizal fungus and its host plant.

BACKGROUND: Small RNAs (sRNAs) are short non-coding RNA molecules (20-30 nt) that regulate gene expression at transcriptional or post-transcriptional levels in many eukaryotic organisms, through a mechanism known as RNA interference (RNAi). Recent studies have highlighted that they are also involved in cross-kingdom communication: sRNAs can move across the contact surfaces from "donor" to "receiver" organisms and, once in the host cells of the receiver, they can target specific mRNAs, leading to a modulation of host metabolic pathways and defense responses. Very little is known about RNAi mechanism and sRNAs occurrence in Arbuscular Mycorrhizal Fungi (AMF), an important component of the plant root microbiota that provide several benefits to host plants, such as improved mineral uptake and tolerance to biotic and abiotic stress. RESULTS: Taking advantage of the available genomic resources for the AMF Rhizophagus irregularis we described its putative RNAi machinery, which is characterized by a single Dicer-like (DCL) gene and an unusual expansion of Argonaute-like (AGO-like) and RNA-dependent RNA polymerase (RdRp) gene families. In silico investigations of previously published transcriptomic data and experimental assays carried out in this work provided evidence of gene expression for most of the identified sequences. Focusing on the symbiosis between R. irregularis and the model plant Medicago truncatula, we characterized the fungal sRNA population, highlighting the occurrence of an active sRNA-generating pathway and the presence of microRNA-like sequences. In silico analyses, supported by host plant degradome data, revealed that several fungal sRNAs have the potential to target M. truncatula transcripts, including some specific mRNA already shown to be modulated in roots upon AMF colonization. CONCLUSIONS: The identification of RNAi-related genes, together with the characterization of the sRNAs population, suggest that R. irregularis is equipped with a functional sRNA-generating pathway. Moreover, the in silico analysis predicted 237 plant transcripts as putative targets of specific fungal sRNAs suggesting that cross-kingdom post-transcriptional gene silencing may occur during AMF colonization.

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.

The arbuscular mycorrhizal symbiosis attenuates symptom severity and reduces virus concentration in tomato infected by Tomato yellow leaf curl Sardinia virus (TYLCSV).

The arbuscular mycorrhizal (AM) symbiosis is considered a natural instrument to improve plant health and productivity since mycorrhizal plants often show higher tolerance to abiotic and biotic stresses. However, the impact of the AM symbiosis on infection by viral pathogens is still largely uncertain and little explored. In the present study, tomato plants were grown under controlled conditions and inoculated with the AM fungus Funneliformis mosseae. Once the mycorrhizal colonization had developed, plants were inoculated with the Tomato yellow leaf curl Sardinia virus (TYLCSV), a geminivirus causing one of the most serious viral diseases of tomatoes in Mediterranean areas. Biological conditions consisted of control plants (C), TYLCSV-infected plants (V), mycorrhizal plants (M), and TYLCSV-infected mycorrhizal plants (MV). At the time of analysis, the level of mycorrhiza development and the expression profiles of mycorrhiza-responsive selected genes were not significantly modified by virus infection, thus indicating that the AM symbiosis was unaffected by the presence and spread of the virus. Viral symptoms were milder, and both shoot and root concentrations of viral DNA were lower in MV plants than in V plants. Overall F. mosseae colonization appears to exert a beneficial effect on tomato plants in attenuating the disease caused by TYLCSV.

Exogenous Application of dsRNA for Protection against Tomato Leaf Curl New Delhi Virus.

Tomato leaf curl New Delhi virus (ToLCNDV) is an emerging plant pathogen, fast spreading in Asian and Mediterranean regions, and is considered the most harmful geminivirus of cucurbits in the Mediterranean. ToLCNDV infects several plant and crop species from a range of families, including Solanaceae, Cucurbitaceae, Fabaceae, Malvaceae and Euphorbiaceae. Up to now, protection from ToLCNDV infection has been achieved mainly by RNAi-mediated transgenic resistance, and non-transgenic fast-developing approaches are an urgent need. Plant protection by the delivery of dsRNAs homologous to a pathogen target sequence is an RNA interference-based biotechnological approach that avoids cultivating transgenic plants and has been already shown effective against RNA viruses and viroids. However, the efficacy of this approach against DNA viruses, particularly Geminiviridae family, is still under study. Here, the protection induced by exogenous application of a chimeric dsRNA targeting all the coding regions of the ToLCNDV DNA-A was evaluated in zucchini, an important crop strongly affected by this virus. A reduction in the number of infected plants and a delay in symptoms appearance, associated with a tendency of reduction in the viral titer, was observed in the plants treated with the chimeric dsRNA, indicating that the treatment is effective against geminiviruses but requires further optimization. Limits of RNAi-based vaccinations against geminiviruses and possible causes are discussed.

Rapid and specific detection of wheat spindle streak mosaic virus using RT-LAMP in durum wheat crude leaf extract.

To accurately determine the spread of any pathogen, including plant viruses, a quick, sensitive, cost-effective, point-of-care diagnostic assay is necessary. Wheat spindle streak mosaic virus (WSSMV) is a Bymovirus, transmitted by the plasmodiophorid Polymyxa graminis Led, which causes yellow mosaic and reduces the grain yield in wheat. Currently, detection protocols for WSSMV use ELISA or more sensitive PCR-based approaches requiring specialized laboratory and personnel. A protocol for reverse transcription loop mediated isothermal amplification (RT-LAMP) has been developed and optimized for the rapid detection of viruses using crude extracts from wheat leaves. The protocol was specific for WSSMV detection, while no reaction was observed with SBCMV or SBWMV, the non-target viruses transmitted by the same vector. The RT-LAMP assay was shown to be as sensitive as the one-step WSSMV specific RT-PCR. The RT-LAMP assay can be performed under field conditions using a portable instrument, and can help the actual spread of WSSMV, an aspect of this virus not yet well understood, to be explored.

Recombination profiles between Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus in laboratory and field conditions: evolutionary and taxonomic implications.

Tomato yellow leaf curl Sardinia virus and Tomato yellow leaf curl virus have co-existed in Italian tomato crops since 2002 and have reached equilibrium, with plants hosting molecules of both species plus their recombinants being the most frequent case. Recombination events are studied in field samples, as well as in experimental co-infections, when recombinants were detected as early as 45 days following inoculation. In both conditions, recombination breakpoints were essentially absent in regions corresponding to ORFs V2, CP and C4, whereas density was highest in the 3'-terminal portion of ORF C3, next to the region where the two transcription units co-terminate. The vast majority of breakpoints were mapped at antisense ORFs, supporting speculation that the rolling-circle replication mechanism, and the existence of sense and antisense ORFs on the circular genome, may result in clashes between replication and transcription complexes.

Fast and Sensitive Detection of Soil-Borne Cereal Mosaic Virus in Leaf Crude Extract of Durum Wheat.

Soil-borne cereal mosaic virus (SBCMV) is a furovirus with rigid rod-shaped particles containing an ssRNA genome, transmitted by Polymyxa graminis Led., a plasmodiophorid that can persist in soil for up to 20 years. SBCMV was reported on common and durum wheat and it can cause yield losses of up to 70%. Detection protocols currently available are costly and time-consuming (real-time PCR) or have limited sensitivity (ELISA). To facilitate an efficient investigation of the real dispersal of SBCMV, it is necessary to develop a new detection tool with the following characteristics: no extraction steps, very fast results, and high sensitivity to allow pooling of a large number of samples. In the present work, we have developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) protocol with such characteristics, and we have compared it with real-time PCR. Our results show that the sensitivity of LAMP and real-time PCR on cDNA and RT-LAMP on crude extracts are comparable, with the obvious advantage that RT-LAMP produces results in minutes rather than hours. This paves the way for extensive field surveys, leading to a better knowledge of the impact of this virus on wheat health and yield.

Identification of Known and Novel Arundo donax L. MicroRNAs and Their Targets Using High-Throughput Sequencing and Degradome Analysis.

MicroRNAs (miRNAs) are a class of non-coding molecules involved in the regulation of a variety of biological processes. They have been identified and characterized in several plant species, but only limited data are available for Arundo donax L., one of the most promising bioenergy crops. Here we identified, for the first time, A. donax conserved and novel miRNAs together with their targets, through a combined analysis of high-throughput sequencing of small RNAs, transcriptome and degradome data. A total of 134 conserved miRNAs, belonging to 45 families, and 27 novel miRNA candidates were identified, along with the corresponding primary and precursor miRNA sequences. A total of 96 targets, 69 for known miRNAs and 27 for novel miRNA candidates, were also identified by degradome analysis and selected slice sites were validated by 5'-RACE. The identified set of conserved and novel candidate miRNAs, together with their targets, extends our knowledge about miRNAs in monocots and pave the way to further investigations on miRNAs-mediated regulatory processes in A. donax, Poaceae and other bioenergy crops.

Arbuscular mycorrhizal symbiosis limits foliar transcriptional responses to viral infection and favors long-term virus accumulation.

Tomato (Solanum lycopersicum) can establish symbiotic interactions with arbuscular mycorrhizal (AM) fungi, and can be infected by several pathogenic viruses. Here, we investigated the impact of mycorrhization by the fungus Glomus mosseae on the Tomato spotted wilt virus (TSWV) infection of tomato plants by transcriptomic and hormones level analyses. In TSWV-infected mycorrhizal plants, the AM fungus root colonization limited virus-induced changes in gene expression in the aerial parts. The virus-responsive upregulated genes, no longer induced in infected mycorrhizal plants, were mainly involved in defense responses and hormone signaling, while the virus-responsive downregulated genes, no longer repressed in mycorrhizal plants, were involved in primary metabolism. The presence of the AM fungus limits, in a salicylic acid-independent manner, the accumulation of abscissic acid observed in response to viral infection. At the time of the molecular analysis, no differences in virus concentration or symptom severity were detected between mycorrhizal and nonmycorrhizal plants. However, in a longer period, increase in virus titer and delay in the appearance of recovery were observed in mycorrhizal plants, thus indicating that the plant's reaction to TSWV infection is attenuated by mycorrhization.

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.

No Evidence for Seed Transmission of Tomato Yellow Leaf Curl Sardinia Virus in Tomato.

Seed transmission is an important factor in the epidemiology of plant pathogens. Geminiviruses are serious pests spread in tropical and subtropical regions. They are transmitted by hemipteran insects, but a few cases of transmission through seeds were recently reported. Here, we investigated the tomato seed transmissibility of the begomovirus tomato yellow leaf curl Sardinia virus (TYLCSV), one of the agents inducing the tomato yellow leaf curl disease, heavily affecting tomato crops in the Mediterranean area. None of the 180 seedlings originating from TYLCSV-infected plants showed any phenotypic alteration typical of virus infection. Moreover, whole viral genomic molecules could not be detected in their cotyledons and true leaves, neither by membrane hybridization nor by rolling-circle amplification followed by PCR, indicating that TYLCSV is not a seed-transmissible pathogen for tomato. Examining the localization of TYLCSV DNA in progenitor plants, we detected the virus genome by PCR in all vegetative and reproductive tissues, but viral genomic and replicative forms were found only in leaves, flowers and fruit flesh, not in seeds and embryos. Closer investigations allowed us to discover for the first time that these embryos were superficially contaminated by TYLCSV DNA but whole genomic molecules were not detectable. Therefore, the inability of TYLCSV genomic molecules to colonize tomato embryos during infection justifies the lack of seed transmissibility observed in this host.

ORTom: a multi-species approach based on conserved co-expression to identify putative functional relationships among genes in tomato.

Co-expressed genes are often expected to be functionally related and many bioinformatics approaches based on co-expression have been developed to infer their biological role. However, such annotations may be unreliable, whereas the evolutionary conservation of gene co-expression among species may form a basis for more confident predictions. The huge amount of expression data (microarrays, SAGE, ESTs) has already allowed functional studies based on conserved co-expression in animals. Up to now, the implementation of analogous tools for plants has been strongly limited probably by the paucity and heterogeneity of data. Here we present ORTom, a tomato-centred EST data-mining approach based on conserved co-expression in the Solanaceae family. ORTom can be used to predict functional relationships among genes and to prioritize candidate genes for targeted studies. The method consists in ranking ESTs co-expressed with a gene of interest according to the level of expression pattern conservation in phylogenetically-related plants (potato, tobacco and pepper) to obtain lists of putative functionally-related genes. The lists are then analyzed for Gene Ontology keyword enrichment. The web server ORTom has been implemented to make the results publicly-available and searchable. Few biological examples on how the tool can be used are presented.

The complete nucleotide sequence of an isolate of Tomato yellow leaf curl Sardinia virus found in Sicily.

Partial sequences of Tomato yellow leaf curl Sardinia virus (TYLCSV) derived from tomato samples collected in Sicily in 1999, 2002 and 2004 indicated the presence of a TYLCSV different from the one previously described as the Sic strain. Here, we report a complete DNA sequence that is classified as belonging to the TYLCSV type strain (Sar strain), confirming the co-existence in Sicily of virus populations of both strains. Moreover, comparisons between this new sequence and those of the two recombinants recently described in Sicily revealed unequivocally (99% identity) that their TYLCSV-derived portion originated from the Sar strain.

The Induction of an Effective dsRNA-Mediated Resistance Against Tomato Spotted Wilt Virus by Exogenous Application of Double-Stranded RNA Largely Depends on the Selection of the Viral RNA Target Region.

Tomato spotted wilt virus (TSWV) is a devastating plant pathogen, causing huge crop losses worldwide. Unfortunately, due to its wide host range and emergence of resistance breaking strains, its management is challenging. Up to now, resistance to TSWV infection based on RNA interference (RNAi) has been achieved only in transgenic plants expressing parts of the viral genome or artificial microRNAs targeting it. Exogenous application of double-stranded RNAs (dsRNAs) for inducing virus resistance in plants, namely RNAi-based vaccination, represents an attractive and promising alternative, already shown to be effective against different positive-sense RNA viruses and viroids. In the present study, the protection efficacy of exogenous application of dsRNAs targeting the nucleocapsid (N) or the movement protein (NSm) coding genes of the negative-sense RNA virus TSWV was evaluated in Nicotiana benthamiana as model plant and in tomato as economically important crop. Most of the plants treated with N-targeting dsRNAs, but not with NSm-targeting dsRNAs, remained asymptomatic until 40 (N. benthamiana) and 63 (tomato) dpi, while the remaining ones showed a significant delay in systemic symptoms appearance. The different efficacy of N- and NSm-targeting dsRNAs in protecting plants is discussed in the light of their processing, mobility and biological role. These results indicate that the RNAi-based vaccination is effective also against negative-sense RNA viruses but emphasize that the choice of the target viral sequence in designing RNAi-based vaccines is crucial for its success.

Arbuscular Mycorrhizal Symbiosis Primes Tolerance to Cucumber Mosaic Virus in Tomato.

Tomato plants can establish symbiotic interactions with arbuscular mycorrhizal fungi (AMF) able to promote plant nutrition and prime systemic plant defenses against pathogens attack; the mechanism involved is known as mycorrhiza-induced resistance (MIR). However, studies on the effect of AMF on viral infection, still limited and not conclusive, indicate that AMF colonization may have a detrimental effect on plant defenses against viruses, so that the term "mycorrhiza-induced susceptibility" (MIS) has been proposed for these cases. To expand the case studies to a not yet tested viral family, that is, Bromoviridae, we investigated the effect of the colonization by the AMF Funneliformis mosseae on cucumber mosaic virus (CMV) infection in tomato by phenotypic, physiological, biochemical, and transcriptional analyses. Our results showed that the establishment of a functional AM symbiosis is able to limit symptoms development. Physiological and transcriptomic data highlighted that AMF mitigates the drastic downregulation of photosynthesis-related genes and the reduction of photosynthetic CO2 assimilation rate caused by CMV infection. In parallel, an increase of salicylic acid level and a modulation of reactive oxygen species (ROS)-related genes, toward a limitation of ROS accumulation, was specifically observed in CMV-infected mycorrhizal plants. Overall, our data indicate that the AM symbiosis influences the development of CMV infection in tomato plants and exerts a priming effect able to enhance tolerance to viral infection.

Comparative analysis of expression profiles in shoots and roots of tomato systemically infected by Tomato spotted wilt virus reveals organ-specific transcriptional responses.

Tomato (Solanum lycopersicon), a model species for the family Solanaceae, is severely affected by Tomato spotted wilt virus (TSWV) worldwide. To elucidate the systemic transcriptional response of plants to TSWV infection, microarray experiments were performed on tomato. Parallel analysis of both shoots and roots revealed organ-specific responses, although the virus was present in similar concentration. In the shoots, genes related to defense and to signal transduction were induced, while there was general repression of genes related to primary and secondary metabolism as well as to amino acid metabolism. In roots, expression of genes involved in primary metabolism and signal transduction appear unaffected by TSWV infection, while those related to the response to biotic stimuli were induced and those associated to the response to abiotic stress were generally repressed or unaltered. Genes related to amino acid metabolism were unaffected, except for those involved in synthesis of secondary compounds, where induction was evident. Differential expression of genes involved in metabolism and response to ethylene and abscisic acid was observed in the two organs. Our results provide new insight into the biology of the economically important interaction between tomato and TSWV.

Two new natural begomovirus recombinants associated with the tomato yellow leaf curl disease co-exist with parental viruses in tomato epidemics in Italy.

Two tomato geminivirus species co-exist in protected crops in Sicily, Tomato yellow leaf curl Sardinia virus (TYLCSV, found in 1989) and Tomato yellow leaf curl virus (TYLCV, found in 2002), and mixed infections have been detected. In a field survey conducted in 2004, the viral intergenic region (IR) was amplified from infected plants, and molecules apparently hybrid between the two species were found, but only in plants where one or both parental species were also present. Two of these hybrids, named 2/2 and 2/5, were isolated and infectious clones were obtained. They were both readily whitefly-transmitted to tomato plants; clone 2/5 produced symptoms typical of TYLCSV and TYLCV, while clone 2/2 produced more severe symptoms, with leaves showing downward curling and rugosity. Sequence analysis showed that both 2/2 and 2/5 are newly generated hybrids, with two recombination sites each. One site, common to both hybrids, is in the stem-loop of the IR. The other is close to the 3'-end of the CP ORF in 2/5 and within the Rep ORF in 2/2. Thus, the 2/2 hybrid virus has a hybrid Rep protein, with the 202 amino-terminal aa from TYLCV and the remaining 155 aa from TYLCSV. Replication assays in leaf disc indicated a lower replicative capacity with respect to parental viruses, a fact that might help to explain why plants infected only by a recombinant have not been found so far.

Global and cell-type gene expression profiles in tomato plants colonized by an arbuscular mycorrhizal fungus.

*Arbuscular mycorrhizal symbiosis develops in roots; extensive cellular reorganizations and specific metabolic changes occur, which are mirrored by local and systemic changes in the transcript profiles. *A TOM2 microarray (c. 12 000 probes) has been used to obtain an overview of the transcriptional changes that are triggered in Solanum lycopersicum roots and shoots, as a result of colonization by the arbuscular mycorrhizal fungus Glomus mosseae. The cell-type expression profile of a subset of genes was monitored, using laser microdissection, to identify possible plant determinants of arbuscule development,. *Microarrays revealed 362 up-regulated and 293 down-regulated genes in roots. Significant gene modulation was also observed in shoots: 85 up- and 337 down-regulated genes. The most responsive genes in both organs were ascribed to primary and secondary metabolism, defence and response to stimuli, cell organization and protein modification, and transcriptional regulation. Six genes, preferentially expressed in arbusculated cells, were identified. *A comparative analysis only showed a limited overlap with transcript profiles identified in mycorrhizal roots of Medicago truncatula, probably as a consequence of the largely nonoverlapping probe sets on the microarray tools used. The results suggest that auxin and abscisic acid metabolism are involved in arbuscule formation and/or functioning.

Arbuscular Mycorrhizal Symbiosis: Plant Friend or Foe in the Fight Against Viruses?

Plant roots establish interactions with several beneficial soil microorganisms including arbuscular mycorrhizal fungi (AMF). In addition to promoting plant nutrition and growth, AMF colonization can prime systemic plant defense and enhance tolerance to a wide range of environmental stresses and below-ground pathogens. A protective effect of the AMF against above-ground pathogens has also been described in different plant species, but it seems to largely rely on the type of attacker. Viruses are obligate biotrophic pathogens able to infect a large number of plant species, causing massive losses in crop yield worldwide. Despite their economic importance, information on the effect of the AM symbiosis on viral infection is limited and not conclusive. However, several experimental evidences, obtained under controlled conditions, show that AMF colonization may enhance viral infection, affecting susceptibility, symptomatology and viral replication, possibly related to the improved nutritional status and to the delayed induction of pathogenesis-related proteins in the mycorrhizal plants. In this review, we give an overview of the impact of the AMF colonization on plant infection by pathogenic viruses and summarize the current knowledge of the underlying mechanisms. For the cases where AMF colonization increases the susceptibility of plants to viruses, the term "mycorrhiza-induced susceptibility" (MIS) is proposed.

Impact of high or low levels of phosphorus and high sodium in soils on productivity and stress tolerance of Arundo donax plants.

The potential of Arundo donax to grow in degraded soils, characterized by excess of salinity (Na+), and phosphorus deficiency (-P) or excess (+P) also coupled with salinity (+NaP), was investigated by combining in vivo plant phenotyping, quantification of metabolites and ultrastructural imaging of leaves with a transcriptome-wide screening. Photosynthesis and growth were impaired by + Na, -P and + NaP. While + Na caused stomatal closure, enhanced biosynthesis of carotenoids, sucrose and isoprene and impaired anatomy of cell walls, +P negatively affected starch production and isoprene emission, and damaged chloroplasts. Finally, +NaP largely inhibited photosynthesis due to stomatal limitations, increased sugar content, induced/repressed a number of genes 10 time higher with respect to + P and + Na, and caused appearance of numerous and large plastoglobules and starch granules in chloroplasts. Our results show that A. donax is sensitive to unbalances of soil ion content, despite activation of defensive mechanisms that enhance plant resilience, growth and biomass production of A. donax under these conditions.