Lateral roots (LRs) increase the contact area of the root with the rhizosphere and thereby improve water and nutrient uptake from the soil. LRs are generated either via a developmentally controlled mechanism or through induction by external stimuli, such as water and nutrient availability. Auxin regulates LR organogenesis via transcriptional activation by an auxin complex receptor. Endocytic trafficking to the vacuole positively regulates LR organogenesis independently of the auxin complex receptor in Arabidopsis (Arabidopsis thaliana). Here, we demonstrate that phosphatidylinositol 4-phosphate (PI4P) biosynthesis regulated by the phosphatidylinositol 4-kinases PI4KIIIb1 and PI4KIIIb2 is essential for the LR organogenesis driven by endocytic trafficking to the vacuole. Stimulation with Sortin2, a biomodulator that promotes protein targeting to the vacuole, altered PI4P abundance at both the plasma membrane and endosomal compartments, a process dependent on PI4K activity. These findings suggest that endocytic trafficking to the vacuole regulated by the enzymatic activities of PI4KIIIb1 and PI4KIIIb2 participates in a mechanism independent of the auxin complex receptor that regulates LR organogenesis in Arabidopsis. Surprisingly, loss-of-function of PI4KIIIb1 and PI4KIIIb2 induced both LR primordium formation and endocytic trafficking toward the vacuole. This LR primordium induction was alleviated by exogenous PI4P, suggesting that PI4KIIIb1 and PI4KIIIb2 activity constitutively negatively regulates LR primordium formation. Overall, this research demonstrates a dual role of PI4KIIIb1 and PI4KIIIb2 in LR primordium formation in Arabidopsis.
Anthracnose caused by Colletotrichum species is one of the most frequent and damaging fungal diseases affecting avocado fruits (Persea americana Mill.) worldwide. In Chile, the disease incidence has increased over the last decades due to the establishment of commercial groves in more humid areas. Since 2018, unusual symptoms of anthracnose have been observed on Hass avocado fruits, with lesions developing a white to gray sporulation. Morphological features and multi-locus phylogenetic analyses using six DNA barcodes (act, chs-1, gapdh, his3, ITS, and tub2) allowed the identification of the causal agent as Colletotrichum anthrisci, a member of the dematium species complex. Pathogenicity was confirmed by inoculating healthy Hass avocado fruits with representative isolates, reproducing the same symptoms initially observed, and successfully reisolating the same isolates from the margin of the necrotic pulp. Previously, several Colletotrichum species belonging to other species complexes have been associated with avocado anthracnose in other countries. To our knowledge, this is the first record of C. anthrisci and of a species of the dematium species complex causing anthracnose on avocado fruits in Chile and worldwide.
Chemical compounds are useful to perturb biological functions in the same way as classical genetic approaches take advantage of mutations at the DNA level to perturb gene function. The use of bioactive chemicals currently called chemical genetic is especially valuable for cell biology. Chemical genetic approaches allow perturbations of cellular processes post-germination in a given time window controlling the severity of the effect by modifying or modulating the dose and/or the period of the treatment. Additionally, compounds can be applied directly to different mutants and translational fluorescent reporters/marker lines, expanding the repertoire of experimental setups addressing cell biology research. In this chapter, we describe standard protocols to visualize vacuole morphology and trafficking to the vacuole and the use of bioactive compounds as a proxy to study these biological processes.
Disease management programmes in Chilean table grape vineyards use the hydroxyanilide fenhexamid as a pivotal fungicide for Botrytis cinerea control. However, fenhexamid-resistant populations of this pathogen have progressively increased in vineyards under fungicide use. Botrytis cinerea isolates were collected in ‘Thompson Seedless’ vineyards under fenhexamid control programmes (>two sprays per season) from three regions of Central Chile, during the 2013-2014, 2014-2015 and 2015-2016 growing seasons. Focusing on the 2015-2016 growing season when the greatest level of resistance was measured, only 8% of recovered isolates were sensitive to fenhexamid with 92% of isolates exceeding the sensitivity threshold for mycelium growth. All fenhexamid resistant isolates analyzed carried a mutation in the Erg27 gene, which encodes for 3-keto reductase (3-KR) enzyme. The largest proportion of isolates presented a single-point mutation, leading to a substitution of phenylalanine by serine or isoleucine in the 412 residue of 3-KR (erg27F412S, 27%; erg27F412I, 48%). Substitution by valine in this position was observed in a lower proportion of isolates (erg27F412V, 2%). In contrast to a previous report indicating high fitness cost in isolates carrying erg27F412S or erg27F412I, mycelium growth and sclerotia development under different restrictive temperatures were not affected compared to wildtype Erg27 F412 in Chilean mutant isolates. At 0°C, erg27F412S and erg27F412I generated larger lesions than erg27F412V and Erg27F412 isolates in wounded and unwounded berry assays. Another five mutations were detected in low-resistance Erg27 F412 isolates; one was a previously unreported mutation: erg27R330P. This study has demonstrated a significant loss of sensitivity to fenhexamid, limited fitness cost and high aggressiveness levels (erg27F412S and erg27 F412I) in field isolates carrying Erg27 mutations, giving dirctions for the design of Botrytis control programmes based on fenhexamid.
Sucrose is a central regulator of plant growth and development, coordinating cell division and cell elongation according to the energy status of plants. Sucrose is known to stimulate bulk endocytosis in cultured cells; however, its physiological role has not been described to date. Our work shows that sucrose supplementation induces root cell elongation and endocytosis. Sucrose targets clathrin-mediated endocytosis (CME) in epidermal cells. Its presence decreases the abundance of both the clathrin coating complex and phosphatidylinositol 4,5-biphosphate at the plasma membrane, while increasing clathrin complex abundance in intracellular spaces. Sucrose decreases the plasma membrane residence time of the clathrin complex, indicating that it controls the kinetics of endocytic vesicle formation and internalization. CME regulation by sucrose is inducible and reversible; this on/off mechanism reveals an endocytosis-mediated mechanism for sensing plant energy status and signaling root elongation. The sucrose monosaccharide fructose also induces CME, while glucose and mannitol have no effect, demonstrating the specificity of the process. Overall, our data show that sucrose can mediate CME, which demonstrates that sucrose signaling for plant growth and development is dependent on endomembrane trafficking.
In Chile, the 2019-2020 sweet cherry season yielded 228,548 t, produced on 38,392 hectares and an average annual crop value about US$1.6 billion (http://www.iqonsulting.com/yb/). Between autumn 2019 and summer of 2020, branch and limbs dieback symptoms were observed in two 12-year-old sweet cherry (Prunus avium L.) orchards located in the O’Higgins region (Chile Central Valley). Furthermore, other symptoms such as wilting leaves, cankers, bark cracking, emission of gum exudates and internal wood necrosis were detected on trees of “Bing”, “Santina” and “Sweetheart” cultivars (Cainelli et al. 2017). Wood fragments from symptomatic branches were surface sterilized with 95% ethanol, flaming and placed onto potato dextrose agar (PDA) amended with 0.5 g liter−1 of streptomycin sulfate (Berbegal et al. 2014). After 7 days of incubation at 25°C, pink to red colonies with white margins were isolated. Each isolate was characterized by having hyaline and oblong-ellipsoidal conidia of 5.76 ± 0.88 × 1.76 ± 0.36 μm (n=100) (Trouillas et al. 2012). According to these morphological features, the fungus was identified as Calosphaeria pulchella (Pers.: Fr.) J. Schröt (anamorph Calosphaeriosphora pulchella Réblová,L. Mostert, W. Gams & Crous) (Réblová et al. 2004). ITS (Internal Transcribed Spacer region of the rDNA) sequence comparison using BLAST analysis revealed a 99.48% identity and 100% query coverage between C. pulchella sequence HM237297 and the Chilean isolates. Moreover, the Chilean isolates were confirmed by means of phylogenetic analysis using ITS sequences of C. pulchella available in GenBank database. The maximum-parsimony phylogenetic tree supported the cluster analysis of the Chilean C. pulchella isolates with those obtained in other regions of the world with a bootstrap value of 95% (Berbegal et al. 2014; Trouillas et al. 2012). The Chilean ITS sequences were deposited into GenBank (MT378444 to MT378447). Two-year-old sweet cherry trees cv. Bing were inoculated with the Chilean isolates. Six trees were used as replicates. To accomplish this goal, two punctures of 5mm diameter were made in two branches per tree with a cork borer and a plug of mycelium from 7-day-old colonies was laid on the wound mycelium side down. Six trees were inoculated with sterile agar plugs. Every puncture was sealed with petroleum jelly and wrapped with parafilm. Four months after inoculation, the vascular streaking developing from the inoculated wounds was measured. The average lesion lengths on inoculated and non-inoculated shoots were 43.79 and 21.79 mm, respectively, which were significantly different according LSD Fisher test (p<0.05). C. pulchella was recovered from all the inoculated branches. No fungus was isolated from the controls, confirming Koch’s postulates (Trouillas et al. 2012). To our knowledge this is the first report of C. pulchella causing canker and branch dieback in sweet cherry trees in Chile. This new disease represents a serious threat to the Chilean cherry industry, and further research on disease control is needed.
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