Silencing of OPR3 in tomato reveals the role of OPDA in callose deposition during the activation of defense responses against Botrytis cinerea

Cis‐(+)‐12‐oxo‐phytodienoic acid (OPDA) is likely to play signaling roles in plant defense that do not depend on its further conversion to the phytohormone jasmonic acid. To elucidate the role of OPDA in Solanum lycopersicum (tomato) plant defense, we have silenced the 12‐oxophytodienoate reductase 3 (OPR3) gene. Two independent transgenic tomato lines (SiOPR3‐1 and SiOPR3‐2) showed significantly reduced OPR3 expression upon infection with the necrotrophic pathogen Botrytis cinerea. Moreover, SiOPR3 plants are more susceptible to this pathogen, and this susceptibility is accompanied by a significant decrease in OPDA levels and by the production of JA‐Ile being almost abolished. OPR3 silencing also leads to a major reduction in the expression of other genes of the jasmonic acid (JA) synthesis and signaling pathways after infection. These results confirm that in tomato plants, as in Arabidopsis, OPR3 determines OPDA availability for JA biosynthesis. In addition, we show that an intact JA biosynthetic pathway is required for proper callose deposition, as its pathogen‐induced accumulation is reduced in SiOPR3 plants. Interestingly, OPDA, but not JA, treatment restored basal resistance to B. cinerea and induced callose deposition in SiOPR3‐1 and SiOPR3‐2 transgenic plants. These results provide clear evidence that OPDA by itself plays a major role in the basal defense of tomato plants against this necrotrophic pathogen.     

Roles of small RNAs in soybean defense against Phytophthora sojae infection

The genus Phytophthora consists of many notorious pathogens of crops and forestry trees. At present, battling Phytophthora diseases is challenging due to a lack of understanding of their pathogenesis. We investigated the role of small RNAs in regulating soybean defense in response to infection by Phytophthora sojae, the second most destructive pathogen of soybean. Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are universal regulators that repress target gene expression in eukaryotes. We identified known and novel small RNAs that differentially accumulated during P. sojae infection in soybean roots. Among them, miR393 and miR166 were induced by heat‐inactivated P. sojae hyphae, indicating that they may be involved in soybean basal defense. Indeed, knocking down the level of mature miR393 led to enhanced susceptibility of soybean to P. sojae; furthermore, the expression of isoflavonoid biosynthetic genes was drastically reduced in miR393 knockdown roots. These data suggest that miR393 promotes soybean defense against P. sojae. In addition to miRNAs, P. sojae infection also resulted in increased accumulation of phased siRNAs (phasiRNAs) that are predominantly generated from canonical resistance genes encoding nucleotide binding‐leucine rich repeat proteins and genes encoding pentatricopeptide repeat‐containing proteins. This work identifies specific miRNAs and phasiRNAs that regulate defense‐associated genes in soybean during Phytophthora infection.     

The C2H2 zinc‐finger protein SlZF3 regulates AsA synthesis and salt tolerance by interacting with CSN5B

Abiotic stresses are a major cause of crop loss. Ascorbic acid (AsA) promotes stress tolerance by scavenging reactive oxygen species (ROS), which accumulate when plants experience abiotic stress. Although the biosynthesis and metabolism of AsA are well established, the genes that regulate these pathways remain largely unexplored. Here, we report on a novel regulatory gene from tomato (Solanum lycopersicum) named SlZF3 that encodes a Cys2/His2‐type zinc‐finger protein with an EAR repression domain. The expression of SlZF3 was rapidly induced by NaCl treatments. The overexpression of SlZF3 significantly increased the levels of AsA in tomato and Arabidopsis. Consequently, the AsA‐mediated ROS‐scavenging capacity of the SlZF3‐overexpressing plants was increased, which enhanced the salt tolerance of these plants. Protein–protein interaction assays demonstrated that SlZF3 directly binds CSN5B, a key component of the COP9 signalosome. This interaction inhibited the binding of CSN5B to VTC1, a GDP‐mannose pyrophosphorylase that contributes to AsA biosynthesis. We found that the EAR domain promoted the stability of SlZF3 but was not required for the interaction between SlZF3 and CSN5B. Our findings indicate that SlZF3 simultaneously promotes the accumulation of AsA and enhances plant salt‐stress tolerance.     

The role of ethylene and wound signaling in resistance of tomato to Botrytis cinerea

Ethylene, jasmonate, and salicylate play important roles in plant defense responses to pathogens. To investigate the contributions of these compounds in resistance of tomato (Lycopersicon esculentum) to the fungal pathogen Botrytis cinerea, three types of experiments were conducted: (a) quantitative disease assays with plants pretreated with ethylene, inhibitors of ethylene perception, or salicylate; (b) quantitative disease assays with mutants or transgenes affected in the production of or the response to either ethylene or jasmonate; and (c) expression analysis of defense-related genes before and after inoculation of plants with B. cinerea. Plants pretreated with ethylene showed a decreased susceptibility toward B. cinerea, whereas pretreatment with 1-methylcyclopropene, an inhibitor of ethylene perception, resulted in increased susceptibility. Ethylene pretreatment induced expression of several pathogenesis-related protein genes before B. cinerea infection. Proteinase inhibitor I expression was repressed by ethylene and induced by 1-methylcyclopropene. Ethylene also induced resistance in the mutant Never ripe. RNA analysis showed that Never ripe retained some ethylene sensitivity. The mutant Epinastic, constitutively activated in a subset of ethylene responses, and a transgenic line producing negligible ethylene were also tested. The results confirmed that ethylene responses are important for resistance of tomato to B. cinerea. The mutant Defenseless, impaired in jasmonate biosynthesis, showed increased susceptibility to B. cinerea. A transgenic line with reduced prosystemin expression showed similar susceptibility as Defenseless, whereas a prosystemin-overexpressing transgene was highly resistant. Ethylene and wound signaling acted independently on resistance. Salicylate and ethylene acted synergistically on defense gene expression, but antagonistically on resistance.     

Overexpression of FBR41 enhances resistance to sphinganine analog mycotoxin‐induced cell death and Alternaria stem canker in tomato

Fumonisin B1 (FB1) and Alternaria alternate f. sp. lycopersici (AAL)‐toxin are classified as sphinganine analog mycotoxins (SAMTs), which induce programmed cell death (PCD) in plants and pose health threat to humans who consume the contaminated crop products. Herein, Fumonisin B1 Resistant41 (FBR41), a dominant mutant allele, was identified by map‐based cloning of Arabidopsis FB1‐resistant mutant fbr41, then ectopically expressed in AAL‐toxin sensitive tomato (Solanum lycopersicum) cultivar. FBR41‐overexpressing tomato plants exhibited less severe cell death phenotype upon AAL‐toxin treatment. Analysis of free sphingoid bases showed that both fbr41 and FBR41‐overexpressing tomato plants accumulated less sphinganine and phytosphingosine upon FB1 and AAL‐toxin treatment, respectively. Alternaria stem canker is a disease caused by AAL and responsible for severe economic losses in tomato production, and FBR41‐overexpressing tomato plants exhibited enhanced resistance to AAL with decreased fungal biomass and less cell death, which was accompanied by attenuated accumulation of free sphingoid bases and jasmonate (JA). Taken together, our results indicate that FBR41 is potential in inhibiting SAMT‐induced PCD and controlling Alternaria stem canker in tomato.     

Brassinosteroids are involved in ethylene‐induced Pst DC3000 resistance in Nicotiana benthamiana

• Plant immunity is regulated by a huge phytohormone regulation network. Ethylene(ET) and brassinosteroids (BRs) play critical roles in plant response to biotic stress; however, the relationship between BR and ET in plant immunity is unclear.
• We used chemical treatments, genetic approaches and inoculation experiments to investigate the relationship between ET and BR in plant defense against Pst DC3000 in Nicotiana benthamiana.
• Foliar applications of ET and BR enhanced plant resistance to Pst DC3000 inoculation, while treatment with brassinazole (BRZ, a specific BR biosynthesis inhibitor) eliminated the ET induced plant resistance to Pst DC3000. Silencing of DWARF 4(DWF4, a key BR biosynthetic gene), BRASSINOSTEROID INSENSITIVE 1 (BRI1, aBR receptor) and BRASSINOSTEROID-SIGNALING KINASE 1 (BSK1, downstream of BRI1) also neutralised the ET‐induced plant resistance to Pst DC3000. ET can induce callose deposition and reactive oxygen species (ROS) accumulation to resistPst DC3000, BRZ‐treated and gene‐silenced were completely eliminate this response.
• Our results suggest BR is involved in ET‐induced plant resistance, the involvement of ET in plant resistance is possibly by the induction of callose deposition and ROS accumulation, in a BR‐dependent manner.

     

The tyrosine‐sulfated peptide receptors PSKR1 and PSY1R modify the immunity of Arabidopsis to biotrophic and necrotrophic pathogens in an antagonistic manner

The tyrosine‐sulfated peptides PSKα and PSY1 bind to specific leucine‐rich repeat surface receptor kinases and control cell proliferation in plants. In a reverse genetic screen, we identified the phytosulfokine (PSK) receptor PSKR1 as an important component of plant defense. Multiple independent loss‐of‐function mutants in PSKR1 are more resistant to biotrophic bacteria, show enhanced pathogen‐associated molecular pattern responses and less lesion formation after infection with the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. By contrast, pskr1 mutants are more susceptible to necrotrophic fungal infection with Alternaria brassicicola, show more lesion formation and fungal growth which is not observed on wild‐type plants. The antagonistic effect on biotrophic and necrotrophic pathogen resistance is reflected by enhanced salicylate and reduced jasmonate responses in the mutants, suggesting that PSKR1 suppresses salicylate‐dependent defense responses. Detailed analysis of single and multiple mutations in the three paralogous genes PSKR1, ‐2 and PSY1‐receptor (PSY1R) determined that PSKR1 and PSY1R, but not PSKR2, have a partially redundant effect on plant immunity. In animals and plants, peptide sulfation is catalyzed by a tyrosylprotein sulfotransferase (TPST). Mutants lacking TPST show increased resistance to bacterial infection and increased susceptibility to fungal infection, mimicking the triple receptor mutant phenotypes. Feeding experiments with PSKα in tpst‐1 mutants partially restore the defense‐related phenotypes, indicating that perception of the PSKα peptide has a direct effect on plant defense. These results suggest that the PSKR subfamily integrates growth‐promoting and defense signals mediated by sulfated peptides and modulates cellular plasticity to allow flexible adjustment to environmental changes.