Rhizobacteria isolated from xerophyte Haloxylon ammodendron manipulate root system architecture and enhance drought and salt tolerance in Arabidopsis thaliana / Las rizobacterias aisladas del xerófito Haloxylon ammodendron manipulan la arquitectura del sistema radicular y mejoran la tolerancia a la sequía y la sal en Arabidopsis thaliana

The objective of this study was to identify bacteria from the rhizosphere of the black saxaul (Haloxylon ammodendron) and test the possibility of using the bacteria for enhancement of drought and/or salt tolerance in the model plant, Arabidopsis thaliana. We collected rhizosphere and bulk soil samples from a natural habitat of H. ammodendron in Iran and identified 58 morphotypes of bacteria that were enriched in the rhizosphere. From this collection, we focused our further experiments on eight isolates. Microbiological analyses showed that these isolates have different levels of tolerance to heat, salt, and drought stresses, and showed different capabilities of auxin production and phosphorous solubilization. We first tested the effects of these bacteria on the salt tolerance of Arabidopsis on agar plate assays. The bacteria substantially influenced the root system architecture, but they were not effective in increasing salt tolerance significantly. Pot assays were then conducted to evaluate the effects of the bacteria on salt or drought tolerance of Arabidopsis on peat moss. Results showed that three of these bacteria (Pseudomonas spp. and Peribacillus sp.) effectively enhanced drought tolerance in Arabidopsis, so that while none of the mock-inoculated plants survived after 19 days of water withholding, the survival rate was 50–100% for the plants that were inoculated with these bacteria. The positive effects of the rhizobacteria on a phylogenetically-distant plant species imply that the desert rhizobacteria may be used to enhance abiotic stress in crops.(AU)

Rhizobacteria isolated from xerophyte Haloxylon ammodendron manipulate root system architecture and enhance drought and salt tolerance in Arabidopsis thaliana.

The objective of this study was to identify bacteria from the rhizosphere of the black saxaul (Haloxylon ammodendron) and test the possibility of using the bacteria for enhancement of drought and/or salt tolerance in the model plant, Arabidopsis thaliana. We collected rhizosphere and bulk soil samples from a natural habitat of H. ammodendron in Iran and identified 58 morphotypes of bacteria that were enriched in the rhizosphere. From this collection, we focused our further experiments on eight isolates. Microbiological analyses showed that these isolates have different levels of tolerance to heat, salt, and drought stresses, and showed different capabilities of auxin production and phosphorous solubilization. We first tested the effects of these bacteria on the salt tolerance of Arabidopsis on agar plate assays. The bacteria substantially influenced the root system architecture, but they were not effective in increasing salt tolerance significantly. Pot assays were then conducted to evaluate the effects of the bacteria on salt or drought tolerance of Arabidopsis on peat moss. Results showed that three of these bacteria (Pseudomonas spp. and Peribacillus sp.) effectively enhanced drought tolerance in Arabidopsis, so that while none of the mock-inoculated plants survived after 19 days of water withholding, the survival rate was 50-100% for the plants that were inoculated with these bacteria. The positive effects of the rhizobacteria on a phylogenetically-distant plant species imply that the desert rhizobacteria may be used to enhance abiotic stress in crops.

Resistance of wheat genotypes to Mycosphaerella graminicola isolates at seedling stage under greenhouse conditions.

One of the most devastating foliar diseases of wheat worldwide is Septoria leaf blotch (STB), caused by Mycosphaerella graminicola (asexual stage/Anamorph: Septoria tritici) which has been recently intensified in some regions in Iran. In this study, 49 wheat genotypes and 20 wheat differential genotypes were evaluated for their reaction to infection by six isolates of M. graminicola collected from infected fields during 2016-2017 at seedling stage under greenhouse conditions. According to the analysis of variance (ANOVA) of leaf pycnidia coverage percentage, a significant difference (p < .01) was observed between M. graminicola isolates and wheat cultivars. The interaction between genotypes and isolates was also significant (p < .01) and the results indicated a specific interaction between genotypes and isolates. The results presented Dezful and West Azerbaijan isolates that were the most virulent with more pathogenesis on differential genotypes. Although 47 of the wheat genotypes were susceptible to all isolates, some genotypes, including Wc-46,224 (Austria), Wc-45,425 (Portugal), Wc-45,565 (Turkey), P.S.No4 (Italy), Dehdasht, M3 Synthetic, KavKaz-k4500, Arina, Flame, and Riband were resistant to all isolates. In addition, the isolates exhibited different virulence patterns on wheat genotypes. The results of this study revealed high virulence of M. graminicola isolates, and Iranian and foreign wheat genotypes, commonly used in the region, presented high susceptibility, and the resistance sources had been identified among genotypes that can be applied in the wheat breeding programs.

ABA spray on Arabidopsis seedlings increases mature plants vigor under optimal and water-deficit conditions partly by enhancing nitrogen assimilation.

Here, we report the effects of a single abscisic acid (ABA) spray on Arabidopsis seedlings on growth, development, primary metabolism, and response to water-deficit stress in adult and next-generation plants. The experiments were performed over 2 years in two different laboratories in Iran and South Africa. In each experiment, fifty 7-day-old Arabidopsis seedlings were sprayed with 10 µM ABA, 1 mM H2 O2 , distilled water, or left without spraying as priming treatments. Water-deficit stress was applied on half of the plants in each treatment by withholding water 2 days after spraying. Results showed that a single ABA spray at the cotyledonary stage significantly increased plant biomass and delayed flowering. The ABA spray significantly enhanced drought tolerance so that the survival rate after rehydration was 100 and 33% in the first and the second experiments, respectively, for ABA-treated plants compared to 35 and 0% for water-sprayed plants. This enhanced drought tolerance was not inheritable. Metabolomics analyses suggested that ABA probably increases the antioxidant capacity of the plant cells and modulates tricarboxylic acid cycle toward enhanced nitrogen assimilation. Strikingly, we also observed that the early water spray decreases mature plant resilience under water-deficit conditions and cause substantial transient metabolomics perturbations.

Microbiome contributes to phenotypic plasticity in saffron crocus.

Saffron crocus is a sterile plant species that propagates vegetatively, and consequently, narrow genetic variation is detected in this species. Besides the narrow genetic variation, there is significant phenotypic variation in different traits in this plant. Here we tested this hypothesis that plant microbiome is a major contributor to the phenotypic variation. We focused our analysis on culturable bacteria that were dominant in saffron fields with high stigma yield compared to the fields with low stigma yield. Following this strategy, four rhizospheric (Cupriavidus metallidurans, Bacillus sp., Solibacillus sp., and Planococcus sp.) and two endophytic bacteria (Serratia oryzae and S. odorifera) were identified. The effects of the bacteria on the growth and development of the model plant Arabidopsis were assessed both in agar plate and pot assays. Results showed that these bacteria influence the vegetative growth and flowering time of Arabidopsis. In the next step, corms of saffron were inoculated with these bacteria and the growth and development of the saffron plants were monitored for five months. Remarkably, inoculation of the bacteria had significant influence on vegetative growth, flowering time, and stigma yield of saffron crocus. Furthermore, one of the bacteria, C. metallidurans, is reported here for the first time as a naturally occurring plant-associated bacteria. Altogether our results suggest that plant microbiome is an important factor in phenotypic variation in saffron crocus.

Beneficial worm allies warn plants of parasite attack below-ground and reduce above-ground herbivore preference and performance.

Antagonistic interactions among different functional guilds of nematodes have been recognized for quite some time, but the underlying explanatory mechanisms are unclear. We investigated responses of tomato (Solanum lycopersicum) to two functional guilds of nematodes-plant parasite (Meloidogyne javanica) and entomopathogens (Heterorhabditis bacteriophora, Steinernema feltiae below-ground, and S. carpocapsae)-as well as a leaf mining insect (Tuta absoluta) above-ground. Our results indicate that entomopathogenic nematodes (EPNs): (1) reduced root knot nematode (RKN) infestation below-ground, (2) reduced herbivore (T. absoluta) host preference and performance above-ground, and (3) induced overlapping plant defence responses by rapidly activating polyphenol oxidase and guaiacol peroxidase activity in roots, but simultaneously suppressing this activity in above-ground tissues. Concurrently, we investigated potential plant signalling mechanisms underlying these interactions using transcriptome analyses. We found that both entomopathogens and plant parasites triggered immune responses in plant roots with shared gene expression. Secondary metabolite transcripts induced in response to the two nematode functional guilds were generally overlapping and showed an analogous profile of regulation. Likewise, we show that EPNs modulate plant defence against RKN invasion, in part, by suppressing active expression of antioxidant enzymes. Inoculations of roots with EPN triggered an immune response in tomato via upregulated phenylpropanoid metabolism and synthesis of protease inhibitors in plant tissues, which may explain decreased egg laying and developmental performance exhibited by herbivores on EPN-inoculated plants. Furthermore, changes induced in the volatile organic compound-related transcriptome indicated that M. javanica and/or S. carpocapsae inoculation of plants triggered both direct and indirect defences. Our results support the hypothesis that plants "mistake" subterranean EPNs for parasites, and these otherwise beneficial worms activate a battery of plant defences associated with systemic acquired resistance and/or induced systemic resistance with concomitant antagonistic effects on temporally co-occurring subterranean plant pathogenic nematodes and terrestrial herbivores.

Virus-induced CRISPR-Cas9 system improved resistance against tomato yellow leaf curl virus.

Plant viruses are the most significant factors associated with massive economical losses in agricultural industries worldwide. Accordingly, many studies are dedicated to making virus-resistant crop varieties each year due to the ever-changing nature of viruses. Recently genome engineering methods have been used to confer interference against eukaryotic viruses. Research results on genome editing technics, in particular, CRISPR-Cas9, promises a feasible solution to make virus-resistant crops. In this research, we explored the possibility of utilizing CRISPR-Cas9 to obtain TYLCV resistant tomato varieties. Moreover, to overcome any potential off-target effects of Cas9, we used an inducible promoter to initiate Cas9 activity in case of the virus attack. Cas9 vector was transformed by the rgsCaM promoter, known as an endogenous silencer of RNAi and overexpressed after a virus attack. The golden gate cloning method was applied to construct sgRNAs. Intergenic region and coat protein-coding sequences of TYLCV were used to design sgRNAs. Infiltrated sensitive Money Maker varieties analyzed by real-time PCR, showed a significant reduction or delayed accumulation of viral DNA compared to the control plants. This result demonstrates the efficiency of using an inducible promoter in CRISPR-Cas9 constructs.

Ethylene and Abscisic Acid Signaling Pathways Differentially Influence Tomato Resistance to Combined Powdery Mildew and Salt Stress.

There is currently limited knowledge on the role of hormones in plants responses to combinations of abiotic and pathogen stress factors. This study focused on the response of tomato near-isogenic lines (NILs) that carry the Ol-1, ol-2, and Ol-4 loci, conferring resistance to tomato powdery mildew (PM) caused by Oidium neolycopersici, to combined PM and salt stress. These NILs were crossed with the notabilis (ABA-deficient), defenceless1 (JA-deficient), and epinastic (ET overproducer) tomato mutants to investigate possible roles of hormone signaling in response to combined stresses. In the NILs, marker genes for hormonal pathways showed differential expression patterns upon PM infection. The epinastic mutation resulted in breakdown of resistance in NIL-Ol-1 and NIL-ol-2. This was accompanied by reduced callose deposition, and was more pronounced under combined salt stress. The notabilis mutation resulted in H2O2 overproduction and reduced susceptibility to PM in NIL-Ol-1 under combined stress, but lead to higher plant growth reduction under salinity and combined stress. Resistance in NIL-ol-2 was compromised by the notabilis mutation, which was potentially caused by reduction of callose deposition. Under combined stress the compromised resistance in NIL-ol-2 was restored. PM resistance in NIL-Ol-4 remained robust across all mutant and treatment combinations. Hormone signaling is critical to the response to combined stress and PM, in terms of resistance and plant fitness. ABA appears to be at the crossroads of disease susceptibility/senescence and plant performance under combined stress These gained insights can aid in narrowing down targets for improving crop performance under stress combinations.

RNAi-mediated down-regulation of SHATTERPROOF gene in transgenic oilseed rape.

Oilseed rape is one of the important oil plants. Pod shattering is one of the problems in oilseed rape production especially in regions with dry conditions. One of the important genes in Brassica pod opening is SHATTERPROOF1 (SHP1). Down-regulation of BnSHP1 expression by RNAi can increase resistance to pod shattering. A 470 bp of the BnSHP1 cDNA sequence constructed in an RNAi-silencing vector was transferred to oilseed rape cv. SLM046. Molecular analysis of T2 transgenic plants by RT-PCR and Real-time PCR showed that expression of the BnSHP alleles was highly decreased in comparison with control plants. Morphologically, transgenic plants were normal and produced seeds at greenhouse conditions. At ripening, stage pods failed to shatter, and a finger pressure was needed for pod opening.

An avirulent tomato powdery mildew isolate induces localized acquired resistance to a virulent isolate in a spatiotemporal manner.

Hypersensitive response (HR) of plant cells to the attack of pathogens induces resistance to subsequent attacks by a broad spectrum of pathogens, leading to acquired resistance. In this study, we characterized the localized acquired resistance (LAR) in the epidermal cells of tomato. First, we report the discovery of a new isolate of tomato powdery mildew occurring in Japan, KTP-02, which has a different virulence spectrum compared with the previously-characterized isolate, KTP-01. Using these two isolates, we investigated LAR phenomenon in the epidermal cells of tomato plants carrying the Ol-4 resistance gene. Ol-4 encodes a nucleotide-binding site leucine-rich repeat protein that triggers HR in the epidermal cells in response to KTP-01 but not KTP-02. We mounted a single conidium of KTP-01 on a single tomato epidermal cell and then monitored the progress of HR in that cell by live microscopy. Once HR occurred in that cell, we mounted a single conidium of KTP-02 on cells adjacent to or at one-cell distance from the first challenged cells, in different time points. With a digital microscope, we consecutively tracked the progress of HR (i.e., induction of LAR) in those cells. Results showed that, in tomato plants carrying the Ol-4 gene, HR to KTP-01 results in induction of HR in the adjacent epidermal cells challenged with KTP-02. Our results show that LAR can be triggered only in adjacent cell layer and lasts 24 to 48 h after HR occurred in the first cell. We did not observe the reverse phenomenon, induced susceptibility to KTP-01 by KTP-02. Altogether, we report an advanced technique for investigating LAR phenomena, and provide data on spatiotemporal characteristics of LAR in tomato epidermal cells.

Linked, if not the same, Mi-1 homologues confer resistance to tomato powdery mildew and root-knot nematodes.

On the short arm of tomato chromosome 6, a cluster of disease resistance (R) genes have evolved harboring the Mi-1 and Cf genes. The Mi-1 gene confers resistance to root-knot nematodes, aphids, and whiteflies. Previously, we mapped two genes, Ol-4 and Ol-6, for resistance to tomato powdery mildew in this cluster. The aim of this study was to investigate whether Ol-4 and Ol-6 are homologues of the R genes located in this cluster. We show that near-isogenic lines (NIL) harboring Ol-4 (NIL-Ol-4) and Ol-6 (NIL-Ol-6) are also resistant to nematodes and aphids. Genetically, the resistance to nematodes cosegregates with Ol-4 and Ol-6, which are further fine-mapped to the Mi-1 cluster. We provide evidence that the composition of Mi-1 homologues in NIL-Ol-4 and NIL-Ol-6 is different from other nematode-resistant tomato lines, Motelle and VFNT, harboring the Mi-1 gene. Furthermore, we demonstrate that the resistance to both nematodes and tomato powdery mildew in these two NIL is governed by linked (if not the same) Mi-1 homologues in the Mi-1 gene cluster. Finally, we discuss how Solanum crops exploit Mi-1 homologues to defend themselves against distinct pathogens.

Write 'systemic small RNAs': read 'systemic immunity'.

About 50 years ago, it was reported that pathogen-infected plants are less susceptible to a broad spectrum of the subsequent pathogen attacks. This form of induced resistance, which resembles the immunisation in mammalian cells, is called systemic acquired resistance (SAR). In the last 10 years, plant molecular biology has been revolutionised by the discovery of RNA silencing, which is also a systemic phenomenon and also contributes to plant immunity. Here, I review these two systemic phenomena in a comparative way to highlight the possibility that systemic silencing contributes to systemic immunity. This potential contribution could be in the process of gene expression reprogramming, which is needed for SAR induction, and/or in SAR signal complex, and/or in establishing SAR in remote tissues and forming priming status.

Identification of tomato phosphatidylinositol-specific phospholipase-C (PI-PLC) family members and the role of PLC4 and PLC6 in HR and disease resistance.

The perception of pathogen-derived elicitors by plants has been suggested to involve phosphatidylinositol-specific phospholipase-C (PI-PLC) signalling. Here we show that PLC isoforms are required for the hypersensitive response (HR) and disease resistance. We characterised the tomato [Solanum lycopersicum (Sl)] PLC gene family. Six Sl PLC-encoding cDNAs were isolated and their expression in response to infection with the pathogenic fungus Cladosporium fulvum was studied. We found significant regulation at the transcriptional level of the various SlPLCs, and SlPLC4 and SlPLC6 showed distinct expression patterns in C. fulvum-resistant Cf-4 tomato. We produced the encoded proteins in Escherichia coli and found that both genes encode catalytically active PI-PLCs. To test the requirement of these Sl PLCs for full Cf-4-mediated recognition of the effector Avr4, we knocked down the expression of the encoding genes by virus-induced gene silencing. Silencing of SlPLC4 impaired the Avr4/Cf-4-induced HR and resulted in increased colonisation of Cf-4 plants by C. fulvum expressing Avr4. Furthermore, expression of the gene in Nicotiana benthamiana enhanced the Avr4/Cf-4-induced HR. Silencing of SlPLC6 did not affect HR, whereas it caused increased colonisation of Cf-4 plants by the fungus. Interestingly, Sl PLC6, but not Sl PLC4, was also required for resistance to Verticillium dahliae, mediated by the transmembrane Ve1 resistance protein, and to Pseudomonas syringae, mediated by the intracellular Pto/Prf resistance protein couple. We conclude that there is a differential requirement of PLC isoforms for the plant immune response and that Sl PLC4 is specifically required for Cf-4 function, while Sl PLC6 may be a more general component of resistance protein signalling.