We report here that all 25 isolates of Tomato yellow leaf curl China virus (TYLCCNV) collected from tobacco, tomato, or Siegesbeckia orientalis plants in different regions of Yunnan Province, China, were associated with DNA␤ molecules. To investigate the biological role of DNA␤, full-length infectious clones of viral DNA and DNA␤ of TYLCCNV isolate Y10 (TYLCCNV-Y10) were agroinoculated into Nicotiana benthamiana, Nicotiana glutinosa, Nicotiana. tabacum Samsun (NN or nn), tomato, and petunia plants. We found that TYLCCNV-Y10 alone could systemically infect these plants, but no symptoms were induced. TYLCCNV-Y10 DNA␤ was required, in addition to TYLCCNV-Y10, for induction of leaf curl disease in these hosts. Similar to TYLCCNV-Y10, DNA␤ of TYLCCNV isolate Y64 was also found to be required for induction of typical leaf curl diseases in the hosts tested. When the ␤C1 gene of TYLCCNV-Y10 DNA␤ was mutated, the mutants failed to induce leaf curl symptoms in N. benthamiana when coinoculated with TYLCCNV-Y10. However, Southern blot hybridization analyses showed that the mutated DNA␤ molecules were replicated. When N. benthamiana and N. tabacum plants were transformed with a construct containing the ␤C1 gene under the control of the Cauliflower mosaic virus 35S promoter, many transgenic plants developed leaf curl symptoms similar to those caused by a virus, the severity of which paralleled the level of ␤C1 transcripts, while transgenic plants transformed with the ␤C1 gene containing a stop codon after the start codon remained symptomless. Thus, expression of a ␤C1 gene is adequate for induction of symptoms of viral infection in the absence of virus.

Eighteen samples of begomoviruses isolated from tobacco, tomato and weed species in Yunnan, China were found to be associated with DNAb molecules, for which the complete nucleotide sequences were found to contain 1333-1355 nt. The 18 DNAb molecules identified consist of three main types, each associated with a different begomovirus species: 72-99 % nucleotide identity was found within one type, but only 39-57 % identity was found between types. All the DNAb molecules reported here and elsewhere contain a 115 nt conserved region that has 93-100 % identity with a consensus sequence, and have a common ORF encoding 118 amino acids on the complementary strand (designated C1). Co-agroinoculation of the DNA-A component of Tomato yellow leaf curl China virus tobacco isolate Y10, with its associated DNAb (Y10b), shows this DNAb to be involved in symptom induction in tobacco and tomato. The in-frame ATG mutation of C1 of Y10b caused much milder symptoms as compared with wild Y10b, indicating a functional role for this ORF. Pairwise nucleotide sequence identity comparisons of DNAb molecules and their cognate viral DNA-A molecules indicate that DNAb molecules have co-evolved with their cognate helper viruses. Recombination between DNAb molecules is documented and a DNAb species concept is proposed and discussed.

Plants use both cell surface-resident pattern recognition receptors (PRRs) and intracellular nucleotide binding leucine-rich repeat (NLR) receptors to detect various pathogens. Plant PRRs typically recognize conserved pathogen-associated molecular patterns (PAMPs) to provide broad-spectrum resistance. By contrast, plant NLRs generally detect pathogen strain-specific effectors and confer race-specific resistance. Here, we demonstrate that the tomato () NLR Sw-5b confers broad-spectrum resistance against American-type tospoviruses by recognizing a conserved 21-amino acid peptide region within viral movement protein NSm (NSm). Sw-5b NB-ARC-LRR domains directly associate with NSm in vitro and in planta. Domain swap, site-directed mutagenesis and structure modeling analyses identified four polymorphic sites in the Sw-5b LRR domain that are critical for the recognition of NSm Furthermore, recognition of NSm by Sw-5b likely disturbs the residues adjacent to R927 in the LRR domain to weaken the intramolecular interaction between LRR and NB-ARC domains, thus translating recognition of NSm into activation of Sw-5b. Natural variation analysis of Sw-5b homologs from wild tomato species of South America revealed that the four polymorphic sites in the Sw-5b LRR domain were positively selected during evolution and are all necessary to confer resistance to tospovirus. The results described here provide a new example of a plant NLR mediating broad-spectrum resistance through recognition of a small conserved PAMP-like region within the pathogen effector.

Vascular entry is a decisive step for the initiation of long-distance movement of infectious and endogenous RNAs, silencing signals and developmental/defense signals in plants. However, the mechanisms remain poorly understood. We used Potato spindle tuber viroid (PSTVd) as a model to investigate the direct role of the RNA itself in vascular entry. We report here the identification of an RNA motif that is required for PSTVd to traffic from nonvascular into the vascular tissue phloem to initiate systemic infection. This motif consists of nucleotides U/C that form a water-inserted cis Watson-Crick/Watson-Crick base pair flanked by short helices that comprise canonical Watson-Crick/Watson-Crick base pairs. This tertiary structural model was inferred by comparison with X-ray crystal structures of similar motifs in rRNAs and is supported by combined mutagenesis and covariation analyses. Hydration pattern analysis suggests that water insertion induces a widened minor groove conducive to protein and/or RNA interactions. Our model and approaches have broad implications to investigate the RNA structural motifs in other RNAs for vascular entry and to study the basic principles of RNA structure-function relationships.

Rice stripe virus (RSV) causes one of the most serious viral diseases of rice (Oryza sativa L.), but the molecular basis of RSV resistance has remained elusive. Here we show that the resistant allele of rice STV11 (STV11-R) encodes a sulfotransferase (OsSOT1) catalysing the conversion of salicylic acid (SA) into sulphonated SA (SSA), whereas the gene product encoded by the susceptible allele STV11-S loses this activity. Sequence analyses suggest that the STV11-R and STV11-S alleles were predifferentiated in different geographic populations of wild rice, Oryza rufipogon, and remained prevalent in cultivated indica and japonica rice varieties, respectively. Introgression of the STV11-R allele into susceptible cultivars or heterologous transfer of STV11-R into tobacco plants confers effective resistance against RSV. Our results shed new insights into plant viral defense mechanisms and suggest effective means of breeding RSV-resistant crops using molecular marker-assisted selection or genetic engineering.