Receptor-like cytoplasmic kinases of different subfamilies differentially regulate SOBIR1/BAK1-mediated immune responses in Nicotiana benthamiana.

Cell-surface receptors form the front line of plant immunity. The leucine-rich repeat (LRR)-receptor-like kinases SOBIR1 and BAK1 are required for the functionality of the tomato LRR-receptor-like protein Cf-4, which detects the secreted effector Avr4 of the pathogenic fungus Fulvia fulva. Here, we show that the kinase domains of SOBIR1 and BAK1 directly phosphorylate each other and that residues Thr522 and Tyr469 of the kinase domain of Nicotiana benthamiana SOBIR1 are required for its kinase activity and for interacting with signalling partners, respectively. By knocking out multiple genes belonging to different receptor-like cytoplasmic kinase (RLCK)-VII subfamilies in N. benthamiana:Cf-4, we show that members of RLCK-VII-6, -7, and -8 differentially regulate the Avr4/Cf-4-triggered biphasic burst of reactive oxygen species. In addition, members of RLCK-VII-7 play an essential role in resistance against the oomycete pathogen Phytophthora palmivora. Our study provides molecular evidence for the specific roles of RLCKs downstream of SOBIR1/BAK1-containing immune complexes.

Immune signaling: receptor-like proteins make the difference.

To resist biotic attacks, plants have evolved a sophisticated, receptor-based immune system. Cell-surface immune receptors, which are either receptor-like kinases (RLKs) or receptor-like proteins (RLPs), form the front line of the plant defense machinery. RLPs lack a cytoplasmic kinase domain for downstream immune signaling, and leucine-rich repeat (LRR)-containing RLPs constitutively associate with the RLK SOBIR1. The RLP/SOBIR1 complex was proposed to be the bimolecular equivalent of genuine RLKs. However, it appears that the molecular mechanisms by which RLP/SOBIR1 complexes and RLKs mount immunity show some striking differences. Here, we summarize the differences between RLP/SOBIR1 and RLK signaling, focusing on the way these receptors recruit the BAK1 co-receptor and elaborating on the negative crosstalk taking place between the two signaling networks.

Fitness penalty in susceptible host is associated with virulence of Soybean mosaic virus on Rsv1-genotype soybean: a consequence of perturbation of HC-Pro and not P3.

The multigenic Rsv1 locus in the soybean plant introduction (PI) 'PI96983' confers extreme resistance against the majority of Soybean mosaic virus (SMV) strains, including SMV-N, but not SMV-G7 and SMV-G7d. In contrast, in susceptible soybean cultivars lacking a functional Rsv1 locus, such as 'Williams82' (rsv1), SMV-N induces severe disease symptoms and accumulates to a high level, whereas both SMV-G7 and SMV-G7d induce mild symptoms and accumulate to a significantly lower level. Gain of virulence by SMV-N on Rsv1-genotype soybean requires concurrent mutations in both the helper-component proteinase (HC-Pro) and P3 cistrons. This is because of the presence of at least two resistance (R) genes, probably belonging to the nucleotide-binding leucine-rich repeat (NB-LRR) class, within the Rsv1 locus, independently mediating the recognition of HC-Pro or P3. In this study, we show that the majority of experimentally evolved mutational pathways that disrupt the avirulence functions of SMV-N on Rsv1-genotype soybean also result in mild symptoms and reduced accumulation, relative to parental SMV-N, in Williams82 (rsv1). Furthermore, the evaluation of SMV-N-derived HC-Pro and P3 chimeras, containing homologous sequences from virulent SMV-G7 or SMV-G7d strains, as well as SMV-N-derived variants containing HC-Pro or P3 point mutation(s) associated with gain of virulence, reveals a direct correlation between the perturbation of HC-Pro and a fitness penalty in Williams82 (rsv1). Collectively, these data demonstrate that gain of virulence by SMV on Rsv1-genotype soybean results in fitness loss in a previously susceptible soybean genotype, this being a consequence of mutations in HC-Pro, but not in P3.

The HC-Pro and P3 cistrons of an avirulent Soybean mosaic virus are recognized by different resistance genes at the complex Rsv1 locus.

The complex Rsv1 locus in soybean plant introduction (PI) 'PI96983' confers extreme resistance (ER) against Soybean mosaic virus (SMV) strain N but not SMV-G7 and SMV-G7d. Both the SMV helper-component proteinase (HC-Pro) and P3 cistrons can serve as avirulence factors recognized by Rsv1. To understand the genetics underlying recognition of the two cistrons, we have utilized two soybean lines (L800 and L943) derived from crosses between PI96983 (Rsv1) and Lee68 (rsv1) with distinct recombination events within the Rsv1 locus. L800 contains a single PI96983-derived member (3gG2) of an Rsv1-associated subfamily of nucleotide-binding leucine-rich repeat (NB-LRR) genes. In contrast, although L943 lacks 3gG2, it contains a suite of five other NB-LRR genes belonging to the same family. L800 confers ER against SMV-N whereas L943 allows limited replication at the inoculation site. SMV-N-derived chimeras containing HC-Pro from SMV-G7 or SMV-G7d gained virulence on L943 but not on L800 whereas those with P3 replacement gained virulence on L800 but not on L943. In reciprocal experiments, SMV-G7- and SMV-G7d-derived chimeras with HC-Pro replacement from SMV-N lost virulence on L943 but retained virulence on L800 whereas those with P3 replacement lost virulence on L800 while remaining virulent on L943. These data demonstrate that distinct resistance genes at the Rsv1 locus, likely belonging to the NB-LRR class, mediate recognition of HC-Pro and P3.

Evaluation of North American isolates of Soybean mosaic virus for gain of virulence on Rsv-genotype soybeans with special emphasis on resistance-breaking determinants on Rsv4.

Resistance to Soybean mosaic virus (SMV) in soybean is conferred by three dominant genes: Rsv1, Rsv3 and Rsv4. Over the years, scientists in the USA have utilized a set of standard pathotypes, SMV-G1 to SMV-G7, to study interaction with Rsv-genotype soybeans. However, these pathotypes were isolated from a collection of imported soybean germplasm over 30 years ago. In this study, 35 SMV field isolates collected in recent years from 11 states were evaluated for gain of virulence on soybean genotypes containing individual Rsv genes. All isolates were avirulent on L78-379 (Rsv1), whereas 19 were virulent on L29 (Rsv3). On PI88788 (Rsv4), 14 of 15 isolates tested were virulent; however, only one was capable of systemically infecting all of the inoculated V94-5152 (Rsv4). Nevertheless, virulent variants from 11 other field isolates were rapidly selected on initial inoculation onto V94-5152 (Rsv4). The P3 cistrons of the original isolates and their variants on Rsv4-genotype soybeans were sequenced. Analysis showed that virulence on PI88788 (Rsv4) was not associated, in general, with selection of any new amino acid, whereas Q1033K and G1054R substitutions were consistently selected on V94-5152 (Rsv4). The role of Q1033K and G1054R substitutions, individually or in combination, in virulence on V94-5152 (Rsv4) was confirmed on reconstruction in the P3 cistron of avirulent SMV-N, followed by biolistic inoculation. Collectively, our data demonstrate that SMV has evolved virulence towards Rsv3 and Rsv4, but not Rsv1, in the USA. Furthermore, they confirm that SMV virulence determinants on V94-5152 (Rsv4) reside on P3.

Amino acid changes in P3, and not the overlapping pipo-encoded protein, determine virulence of soybean mosaic virus on functionally immune Rsv1-genotype soybean.

A small open reading frame, termed 'pipo', is embedded in the P3 cistron of potyviruses. Currently, knowledge on pipo and its role(s) in the life cycle of potyviruses is limited. The P3 and helper-component proteinase (HC-Pro) cistrons of Soybean mosaic virus (SMV) harbour determinants affecting virulence on functionally immune Rsv1-genotype soybeans. Interestingly, a key virulence determinant of SMV on Rsv1-genotype soybeans (i.e. soybeans containing the Rsv1 resistance gene) that resides at polyprotein codon 947 overlaps both P3 and a pipo-encoded codon. This raises the question of whether PIPO or P3 is the virulence factor. To answer this question, the corresponding pipo of an avirulent and two virulent strains of SMV were studied by comparative genomics, followed by syntheses and analyses of site-directed mutants. Our data demonstrate that the virulence of SMV on Rsv1-genotype soybeans is affected by P3 and not the overlapping pipo-encoded protein.

Molecular characterization of a new Tospovirus infecting soybean.

A new, widespread disease was recently observed in soybean in the United States. The disease, named Soybean vein necrosis, is manifested by intraveinal chlorosis and necrosis, and has been found in almost all of the 50 fields visited over a period of 3 years in the midwest and midsouth part of the United States. A virus was isolated from symptomatic material, and detection protocols were developed. More than 150 symptomatic specimens collected from seven US States were tested, and all were found positive for the virus unlike 75 asymptomatic samples, revealing the absolute association between virus and disease. Protein pairwise comparisons coupled with phylogenetic analyses indicate that the virus is a new member of the genus Tospovirus.

Experimental adaptation of an RNA virus mimics natural evolution.

Identification of virulence determinants of viruses is of critical importance in virology. In search of such determinants, virologists traditionally utilize comparative genomics between a virulent and an avirulent virus strain and construct chimeras to map their locations. Subsequent comparison reveals sequence differences, and through analyses of site-directed mutants, key residues are identified. In the absence of a naturally occurring virulent strain, an avirulent strain can be functionally converted to a virulent variant via an experimental evolutionary approach. However, the concern remains whether experimentally evolved virulence determinants mimic those that have evolved naturally. To provide a direct comparison, we exploited a plant RNA virus, soybean mosaic virus (SMV), and its natural host, soybean. Through a serial in vivo passage experiment, the molecularly cloned genome of an avirulent SMV strain was converted to virulent variants on functionally immune soybean genotypes harboring resistance factor(s) from the complex Rsv1 locus. Several of the experimentally evolved virulence determinants were identical to those discovered through a comparative genomic approach with a naturally evolved virulent strain. Thus, our observations validate an experimental evolutionary approach to identify relevant virulence determinants of an RNA virus.

Mutational analysis of the putative pipo of soybean mosaic virus suggests disruption of PIPO protein impedes movement.

The presence of a small open reading frame embedded in the P3 cistron of potyvirus turnip mosaic virus, termed "pipo," was recently discovered. We have now studied the putative pipo of soybean mosaic virus (SMV). Introduction of single, or multiple, stop codon mutations at different locations within pipo, without substitution in polyprotein amino acids, did not abolish replication, but restricted the virus to small cluster of cells within the inoculated leaves. Furthermore, extensive mutagenesis of the conserved GA(6) motif at the 5' end of pipo also generated two out of five mutants that remained restricted to small foci of infected cells within the inoculated leaves. Long-distance movement function of the movement-defective PIPO-mutants was not restored following co-inoculation with competent SMV strains. Taken together, the data suggest that the putative pipo of SMV is essential for the virus movement; however, knock out of its expression does not abolish replication.

Occurrence of Alfalfa mosaic virus in Soybean in Tennessee.

Alfalfa mosaic virus (AMV), a member of the genus Alfamovirus in the family Bromoviridae, naturally infects a wide range of plant species (1). Soybean (Glycine max (L.) Merr.) has seldom been reported as a natural host of AMV and there are limited reports of detection of AMV in field-grown soybean plants (4). However, AMV incidence in soybean fields in the midwestern United States has been on the rise in recent years, which is partly attributed to the introduction of the soybean aphid (Aphis glycines) (1,4). In June 2009, soybean plants of cv. Lee68 exhibiting moderate leaf distortion, mottling, and stunting were observed at the East Tennessee Research and Education Center. Leaf samples from 18 symptomatic plants were collected and the sap was extracted and analyzed by antigen-coated indirect ELISA (3) with polyclonal antibodies against AMV, Soybean mosaic virus (SMV), and Bean pod mottle virus (BPMV). None of the samples tested positive for BPMV, but all were found to be infected with SMV. Sap extract from 1 of 18 plants tested positive for AMV and SMV. Sap from this infected plant ground in 10 mM phosphate buffer, pH 7.0, was mechanically inoculated to Carborundum-dusted unifoliate leaves of PI96983, which contains the dominant Rsv1-locus conferring functional immunity to a majority of SMV strains (2). AMV, not SMV, was detected by ELISA in the systemically infected trifoliolate leaves that exhibited moderate mottling, mild leaf distortion, and stunting 14 days postinoculation. Sap was extracted from the infected tissues and the virus was passaged four times through PI96983 before being inoculated to Phaseolus vulgaris cv. Blue Lake. A local lesion isolate was obtained following three successive passages in this host and the isolate was propagated in soybean cv. Williams82. The biologically purified isolate was capable of infecting soybean cvs. L78-379 (Rsv1), L81-4420 (Rsv1), L29 (Rsv3), V94-5152 (Rsv4), Lee68, and Colfax upon sap inoculation. The infected plants exhibited a range of systemic symptoms including mottling, leaf distortion, necrosis, chlorosis, and moderate stunting. To characterize the virus further, total RNA was extracted from infected Williams82 leaf tissues with the RNeasy Plant Mini Kit (Qiagen, Valencia, CA). The RNA served as a template for cDNA synthesis in the presence of random primers. The resultant cDNA served as a template in a PCR assay with primers 1193 (forward) (5'-AGCTGAATTCATGAGTTCTTCACAAC-3') and 1858 (reverse) (5'-GCTAGCGGCCGCTCAATGACGATC-3') corresponding to nucleotides 1,193 to 1,210 and 1,858 to 1,840 of RNA3 from AMV-Kr (GenBank Accession No. AB126032), respectively. The amplified fragments were purified and directly sequenced bidirectionally using the same primers. BLAST analysis of the resultant nucleotide sequences showed 98% identity to an AMV isolate from a naturally infected soybean plant in Illinois (GenBank Accession No. HQ185569), and 97% identity to an isolate described from P. vulgaris in the United States (GenBank Accession No. AY340070.1). To our knowledge, this is the first report of natural infection of soybean by AMV in Tennessee. References: (1) J. Bol. Mol. Plant Pathol. 4:1, 2003. (2) M. R. Hajimorad and J. H. Hill. Mol. Plant-Microbe Interact. 14:587, 2001. (3) M. Malapi-Nelson et al. Plant Dis. 93:1259, 2009. (4) E. E. Mueller and C. R. Grau. Plant Dis. 91:266, 2007.

Genetic variability and phylogenetic analysis of hosta virus X.

Triple gene block 1 (TGB1) and coat protein (CP) sequences of 30 hosta virus X (HVX) isolates from Tennessee (TN), USA, were determined and compared with available sequences in GenBank. The CPs of all known HVX isolates, including those from TN, shared 98.3-100% and 98.2-100% nucleotide and amino acid sequence identity, respectively, whereas TGB1 shared 97.4-100% nucleotide and 97-100% amino acid sequence identity. TGB1 of TN isolates were all longer by one codon from that of a Korean isolate, which is the only sequence publicly available. Phylogenetic analysis of nucleotide and amino acid sequences of TGB1 and CP of all known HVX isolates, separately or combined, revealed a close relationship, suggesting that all of them are derived from a common ancestor. Phylogenetic analysis with the type member of each genus of the family Flexiviridae confirmed that HVX is a member of a distinct species of the genus Potexvirus.

Co-infection of Soybean with Soybean mosaic virus and Alfalfa mosaic virus Results in Disease Synergism and Alteration in Accumulation Level of Both Viruses.

Co-infection of potyviruses with taxonomically diverse plant viruses results in disease synergism and elevation in the level of accumulation of non-potyviruses involved. In the majority of cases, however, the accumulation level of potyviruses remains essentially unaltered. A few potyviruses, such as Soybean mosaic virus (SMV), naturally infect soybean (Glycine max). Soybean is also a natural host to a number of non-potyviruses including Alfalfa mosaic virus (AMV), which causes mild symptoms often associated with symptom remission. We have now studied the interactions between AMV and SMV on symptom severity and accumulation level of each of the two viruses in soybean. Co-infection of soybean with AMV and SMV was established following mechanical inoculation, irrespective of simultaneous or sequential introduction of the two viruses. In multiple experiments, co-infection of soybean resulted in severe symptoms in doubly infected plants in a strain-independent manner, with enhancement in the level of AMV indicating that the interaction of AMV with SMV is synergistic. Conversely, the level of SMV accumulation was reduced. This suggests that in co-infection with AMV, SMV interacts antagonistically. The observation that co-infection of AMV and SMV results in disease synergism suggests enhancement of potential that AMV may become a serious viral disease of soybean.