Root endophytic fungus Serendipita indica modulates barley leaf blade proteome by increasing the abundance of photosynthetic proteins in response to salinity.

AIMS: The present study aimed at analysing the proteome pattern of the leaf blade of barley (Hordeum vulgare L.) in Serendipita indica-colonised plants to decipher the molecular mechanism of S. indica-mediated salt stress. This work is aligned with our previous research on barley leaf sheath to study proteomic pattern variability in leaf blade and sheath of barley plant in response to salinity and S. indica inoculation. METHODS AND RESULTS: The experiment was conducted using a completely randomised factorial design with four replications and two treatments: salinity (0 and 300 mmol l-1 NaCl) and fungus (noninoculated and S. indica-inoculated). The leaf blades of the salt-treated S. indica-colonised and noninoculated plants were harvested after 2 weeks of salt treatment for the physiological and proteomic analyses. After exposure to 300 mmol l-1 NaCl, shoot dry matter production in noninoculated control plants decreased 84% which was about twofold higher than inoculated plants with S. indica. However, the accumulation of sodium in the shoot of S. indica-inoculated plants was significantly lower than the control plants. Analysis of the proteome profile revealed a high number of significantly up-regulated proteins involved in photosynthesis (26 out of 42 identified proteins). CONCLUSIONS: The results demonstrated how the enhanced plant growth and salt stress resistance induced by S. indica was positively associated with the up-regulation of several proteins involved in photosynthesis and carbohydrate metabolism. In fact, S. indica improved photosynthesis in order to reach the best possible performance of the host plant under salt stress. SIGNIFICANCE AND IMPACT OF THE STUDY: Current research provides new insight into the mechanism applied by S. indica in reducing the negative impacts of salt stress in barley at physiological and molecular levels.

Diversity and abundance of culturable nitrogen-fixing bacteria in the phyllosphere of maize.

AIMS: The present study aimed at gaining an insight into the abundance and genetic diversity of culturable N-fixing epiphyte bacteria on the phyllosphere of maize in arid and semi-arid regions of Iran. METHODS AND RESULTS: Leaf samples of the maize variety, 'single cross 704' (Zea mays L.) were collected from different locations in Iran. The community of culturable N-fixing epiphyte bacteria present was examined by 16S rRNA sequencing, BOXAIR-polymerase chain reaction (PCR) and restricted fragment length polymorphisms analysis of 16S rRNA gene (16S-RFLP). Approximately, 31·82% of the 242 isolates were identified as N-fixers by cultivation of bacteria in Rennie medium and detection of their nifH gene. The N-fixers were affiliated with four bacterial phyla: Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes. 16S rRNA sequencing detected 16 genera and 24 different species in the identified phyla. The most dominant genus was Bacillus and the species identified were B. pumilus, B. amyloliquefaciens, B. subtilis, B. paralicheniformis, B. licheniformis, B. niabensis and B. megaterium. In total, 22 RFLP groups were present among the isolates originally identified as N-fixing bacteria. BOXAIR-PCR showed that there was a low similarity level among the N-fixing bacteria isolates, and genetic differentiation of individual strains was relatively great. CONCLUSIONS: Our findings suggest that nitrogen-fixing epiphyte bacteria on the phyllosphere of maize may provide significant nitrogen input into arid and semi-arid ecosystem. SIGNIFICANCE AND IMPACT OF THE STUDY: This research implies that phyllosphere epiphyte diazotrophs have much to offer in sustainable agriculture and can be an alternative to chemical N-fertilizers for providing nitrogen to crops arid and semi-arid regions.

Amino acid substitution in P3 of Soybean mosaic virus to convert avirulence to virulence on Rsv4-genotype soybean is influenced by the genetic composition of P3.

The modification of avirulence factors of plant viruses by one or more amino acid substitutions converts avirulence to virulence on hosts containing resistance genes. Limited experimental studies have been conducted on avirulence/virulence factors of plant viruses, in particular those of potyviruses, to determine whether avirulence/virulence sites are conserved among strains. In this study, the Soybean mosaic virus (SMV)-Rsv4 pathosystem was exploited to determine whether: (i) avirulence/virulence determinants of SMV reside exclusively on P3 regardless of virus strain; and (ii) the sites residing on P3 and crucial for avirulence/virulence of isolates belonging to strain G2 are also involved in virulence of avirulent isolates belonging to strain G7. The results confirm that avirulence/virulence determinants of SMV on Rsv4-genotype soybean reside exclusively on P3. Furthermore, the data show that sites involved in the virulence of SMV on Rsv4-genotype soybean vary among strains, with the genetic composition of P3 playing a crucial role.

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.

Diagnostic Potential of Polyclonal Antibodies Against Bacterially Expressed Recombinant Coat Protein of Alfalfa mosaic virus.

Alfalfa mosaic virus (AMV), a pathogen of a wide range of plant species, including Glycine max (soybean), is poorly immunogenic. Polyclonal antibodies were generated against bacterially expressed recombinant coat proteins (rCPs) of two biologically distinct AMV strains in rabbits and compared with those raised against native and glutaraldehyde-treated virions of the same strains. Analyses showed that sera against rCPs had comparable antibody titers in indirect enzyme-linked immunosorbent assay with those raised against virions when soybean sap containing homologous viruses served as antigens. Polyclonal antibodies against rCPs were specific, sensitive, and detected all AMV isolates that originated from soybean fields from geographically different regions of the United States. Comparison of CP genes of these isolates showed 96 to 99 and 96 to 100% nucleotide and amino acid sequence identities, respectively, suggesting that they are all closely related. This was further confirmed by phylogenetic analysis where they were all clustered together along with representative AMV strains belonging to group I. Collectively, our data demonstrate that, despite poor immunogenicity of AMV, polyclonal antibodies against rCP are effective probes for detection and diagnosis of the virus.