Arabidopsis inositol polyphosphate kinases IPK1 and ITPK1 modulate crosstalk between SA-dependent immunity and phosphate-starvation responses.

KEY MESSAGE: Selective Arabidopsis thaliana inositol phosphate kinase functions modulate response amplitudes in innate immunity by balancing signalling adjustments with phosphate homeostasis networks. Pyrophosphorylation of InsP6 generates InsP7 and/or InsP8 containing high-energy phosphoanhydride bonds that are harnessed during energy requirements of a cell. As bona fide co-factors for several phytohormone networks, InsP7/InsP8 modulate key developmental processes. With requirements in transducing jasmonic acid (JA) and phosphate-starvation responses (PSR), InsP8 exemplifies a versatile metabolite for crosstalks between different cellular pathways during diverse stress exposures. Here we show that Arabidopsis thaliana INOSITOL PENTAKISPHOSPHATE 2-KINASE 1 (IPK1), INOSITOL 1,3,4-TRISPHOSPHATE 5/6-KINASE 1 (ITPK1), and DIPHOSPHOINOSITOL PENTAKISPHOSPHATE KINASE 2 (VIH2) implicated in InsP8 biosynthesis, suppress salicylic acid (SA)-dependent immunity. In ipk1, itpk1 or vih2 mutants, constitutive activation of defenses lead to enhanced resistance against the Pseudomonas syringae pv tomato DC3000 (PstDC3000) strain. Our data reveal that upregulated SA-signaling sectors potentiate increased expression of several phosphate-starvation inducible (PSI)-genes, previously known in these mutants. In reciprocation, upregulated PSI-genes moderate expression amplitudes of defense-associated markers. We demonstrate that SA is induced in phosphate-deprived plants, however its defense-promoting functions are likely diverted to PSR-supportive roles. Overall, our investigations reveal selective InsPs as crosstalk mediators in defense-phosphate homeostasis and in reprogramming stress-appropriate response intensities.

Global SUMOylome Adjustments in Basal Defenses of Arabidopsis thaliana Involve Complex Interplay Between SMALL-UBIQUITIN LIKE MODIFIERs and the Negative Immune Regulator SUPPRESSOR OF rps4-RLD1.

Steady-state SUMOylome of a plant is adjusted locally during developmental transitions and more globally during stress exposures. We recently reported that basal immunity in Arabidopsis thaliana against Pseudomonas syringae pv tomato strain DC3000 (PstDC3000) is associated with strong enhancements in the net SUMOylome. Transcriptional upregulations of SUMO conjugases, suppression of protease, and increased SUMO translations accounted for this enhanced SUMOylation. Antagonistic roles of SUMO1/2 and SUMO3 isoforms further fine-tuned the SUMOylome adjustments, thus impacting defense amplitudes and immune outcomes. Loss of function of SUPPRESSOR OF rps4-RLD1 (SRFR1), a previously reported negative regulator of basal defenses, also caused constitutive increments in global SUMO-conjugates through similar modes. These suggest that SRFR1 plays a pivotal role in maintenance of SUMOylation homeostasis and its dynamic changes during immune elicitations. Here, we demonstrate that SRFR1 degradation kinetically precedes and likely provides the salicylic acid (SA) elevations necessary for the SUMOylome increments in basal defenses. We show that SRFR1 not only is a SUMOylation substrate but also interacts in planta with both SUMO1 and SUMO3. In sum1 or sum3 mutants, SRFR1 stabilities are reduced albeit by different modes. Whereas a srfr1 sum1 combination is lethal, the srfr1 sum3 plants retain developmental defects and enhanced immunity of the srfr1 parent. Together with increasing evidence of SUMOs self-regulating biochemical efficiencies of SUMOylation-machinery, we present their impositions on SRFR1 expression that in turn counter-modulates the SUMOylome. Overall, our investigations reveal multifaceted dynamics of regulated SUMOylome changes via SRFR1 in defense-developmental balance.

Apple scar skin viroid naked RNA is actively transmitted by the whitefly Trialeurodes vaporariorum.

Nucleic acid transfer between plants is a phenomenon which is likely to occur in many ways in nature. We report here the active transmission of Apple scar skin viroid (ASSVd) naked ssRNA species by the whitefly Trialeurodes vaporariorum (Tv). Not only the viroid RNA, its DNA form was also identified from the insect. The viroid transfer efficiency was enhanced with the help of Cucumis sativus Phloem protein 2 (CsPP2), a plant protein known to translocate viroid RNAs. This PP2/ASSVd complex is stably present in the viroid infected cucumber plants, as was identified with the help of immunological reaction. As viroid-like secondary structures are found in some plant RNAs, and PP2 is known to bind and translocate several RNAs, the results have huge implications in transfer of these RNA species between plants visited by the whitefly.

A simplified strategy for studying the etiology of viral diseases: Apple stem grooving virus as a case study.

A simple method to amplify infective, complete genomes of single stranded RNA viruses by long distance PCR (LD PCR) from woody plant tissues is described in detail. The present protocol eliminates partial purification of viral particles and the amplification is achieved in three steps: (i) easy preparation of template RNA by incorporating a pre processing step before loading onto the column (ii) reverse transcription by AMV or Superscript reverse transcriptase and (iii) amplification of cDNA by LD PCR using LA or Protoscript Taq DNA polymerase. Incorporation of a preprocessing step helped to isolate consistent quality RNA from recalcitrant woody tissues such as apple, which was critical for efficient amplification of the complete genomes of Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV). Complete genome of ASGV was cloned under T7 RNA polymerase promoter and was confirmed to be infectious through transcript inoculation producing symptoms similar to the wild type virus. This is the first report for the largest RNA virus genome amplified by PCR from total nucleic acid extracts of woody plant tissues.